Ifngr2 binding molecules and methods of use

ABSTRACT

The present disclosure relates to biologically active molecules comprising a single domain antibody (sdAb) that specifically binds to the extracellular domain of human IFNgR2, compositions comprising such antibodies, and methods of use thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a 371 National Phase Application ofPCT/US2021/044610, filed Aug. 5, 2021, which application claims priorityto U.S. Provisional Application No. 63/061,562, filed Aug. 5, 2020, U.S.Provisional Application No. 63/078,745, filed Sep. 15, 2020, and U.S.Provisional Application No. 63/135,884, filed Jan. 11, 2021, thedisclosures of which are hereby incorporated by reference in theirentirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 31, 2021, isnamed 106249-1258372_SL.txt and is 99,045 bytes in size.

FIELD OF THE INVENTION

The present disclosure relates to biologically active moleculescomprising a single domain antibody that specifically binds to theextracellular domain of the Interferon-γ R2 (IFNgR2), compositionscomprising such single domain antibodies, and methods of use thereof.

BACKGROUND

Interferon gamma is homodimer of two 17 kDa subunits. The IFN-γ receptoris tetramer comprising two ligand-binding IFNγR1 subunits whichassociated two signal-transducing IFNγR2 subunits. IFN-γ binding resultsin the oligomerization of the intracellular domains of IFNγR2 andactivation of intracellular signaling vi JAK1 and JAK2. Phosphorylationof the intracellular domain of the IFNγR2 creates binding sites forSTAT1 which is in turn phosphorylated which dimerize and translocate tothe nucleus. The IFNγ signaling pathway results in a variety ofbiological responses primarily associated with host defense and immunesurveillance. IFNγ results upregulation of the major histocompatibilitycomplex (MHC) molecules as well as the upregulation MHC I and II antigenprocessing and presentation machinery.

IFN-γ is a major product of Th1-mediated immune response. Upregulationof cell surface MHC class I by IFN-γ is an essential element in theresponse to intracellular pathogens and tumor cells. IFN-γ acts as acytotoxic CD8 T cell differentiation signal and is essential for CD8Tcell proliferation. IFN-γ also upregulates cell surface MHC class II onantigen presenting cells and activation of CD4 T cells and activates.The central role of IFN-γ in the immune response can be illustrated byits involvement in more 290 genes related to cytokine and chemokinereceptors, cell activation markers, cellular adhesion proteins, MHCproteins, proteasome formation, protein turnover, and signalingmediators and regulators.

As a result of the central role of IFNγ in the immune response,inhibition of IFNγ is associated with significant downregulation ofimmunity and morbidity. IFNγR1 deficiency is a life-threateningautosomal recessive immune disorder and affected children frequently dieof infection in the absence of bone marrow transplantation. Affectedchildren invariably die of mycobacterial infection, unless bone marrowtransplantation is undertaken. Even then, survivors are susceptible tosevere infection from environmental factors.

Although monoclonal antibodies are the most widely used reagents for thedetection and quantification of proteins, monoclonal antibodies arelarge molecules of about 150 kDa and it sometimes limits their use inassays with several reagents competing for close epitopes recognition. Aunique class of immunoglobulin containing a heavy chain domain andlacking a light chain domain (commonly referred to as heavy chain”antibodies (HCAbs) is present in camelids, including dromedary camels,Bactrian camels, wild Bactrian camels, llamas, alpacas, vicuñas, andguanacos as well as cartilaginous fishes such as sharks. The isolatedvariable domain region of HCAbs is known as a VHH (an abbreviation for“variable-heavy-heavy” reflecting their architecture) or Nanobody®(Ablynx). Single domain VHH antibodies possesses the advantage of smallsize (˜12-14 kD), approximately one-tenth the molecular weight aconventional mammalian IgG class antibody) which facilitates the bindingof these VHH molecules to antigenic determinants of the target which maybe inaccessible to a conventional monoclonal IgG format (Ingram et al.,2018). Furthermore, VHH single domain antibodies are frequentlycharacterized by high thermal stability facilitating pharmaceuticaldistribution to geographic areas where maintenance of the cold chain isdifficult or impossible. These properties, particularly in combinationwith simple phage display discovery methods that do not requireheavy/light chain pairing (as is the case with IgG antibodies) andsimple manufacture (e.g., in bacterial expression systems) make VHHsingle domain antibodies useful in a variety of applications includingthe development of imaging and therapeutic agents.

SUMMARY OF THE INVENTION

The present disclosure provides polypeptides that specifically bind tothe extracellular domain of IFNgR2.

The present disclosure provides a IFNgR2 binding molecule thatspecifically bind to the extracellular domain of IFNgR2 (e.g., humanIFNgR2).

In some embodiments, the IFNgR2 binding molecule comprises a singledomain antibody (sdAb) that specifically binds to the extracellulardomain of the human IFNgR2.

In some embodiments, the IFNgR2 binding molecule is a sdAb, the sdAbcomprising a set of CDRs corresponding to CDR1, CDR2, and CDR3 as shownin a row of Table 1 below.

In some embodiments, the IFNgR2 binding molecule comprises a CDR1, aCDR2, and a CDR3 as described in a row of Table 1 below, in which theCDR1, CDR2, and CDR3 can each, independently, comprise at least 90%(e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequenceidentity, or have 0, 1, 2, or 3 amino acid changes, optionallyconservative amino acid changes, relative to the sequence described in arow of Table 1 below.

In some embodiments, the IFNgR2 binding molecule consists of, optionallyconsists essentially of, or optionally comprises a single domainantibody (sdAb) having at least 80%, alternatively at least 85%,alternatively at least 90%, alternatively at least 95%, alternatively atleast 98%, alternatively at least 99% identity (or being identicalexcept for 1, 2, 3, or 4 amino acids that optionally are conservedsubstitutions) or 100% identity to a polypeptide sequence of any one ofSEQ ID NOS: 2-33, as shown in Table 1 below.

TABLE 1 IFNgR3 VHHs and CDRs Name Sequence (CDRs underlined) CDR1 CDR2CDR3 DR927 QVQLVESGGGLVQAGGSLRL RTFTSYAMN AISWSSGNT TTIATMSDEYTSCAASGRTFTSYAMNWFRQA (SEQ ID NO: 34) YVADSVKG Y (SEQ IDPGKEREFVAAISWSSGNTYVA (SEQ ID NO: 36) DSVKGRFAISRDKAKNTMYL NO: 35)QMNSLAPEDTAVYYCAATTI ATMSDEYTYWGQGTQVTVSS (SEQ ID NO: 2) DR928QVQLVESGGGLVQAGGSLRL RTFSNYRMG AISRGGGTT GDFSTTWDEYNSCAASGRTFSNYRMGWFRQA (SEQ ID NO: 37) LYADSVKG Y PGKEREFVAAISRGGGTTLYA(SEQ ID (SEQ ID NO: 39) DSVKGRFTISRDNAKNTVDLQ NO: 38)MNRLKPEDTAVYFCAAGDFS TTWDEYNYWGQGTQVTVSS (SEQ ID NO: 3) DR929QVQLVESGGGLVQAGGSLRL RTFSSYAMG AISRGGGST RAYSGRYYQFLSCVASGRTFSSYAMGWFRQT (SEQ ID NO: 40) DYADSVKG EYDY PGKEREFVSAISRGGGSTDYA(SEQ ID (SEQ ID NO: 42) DSVKGRFTISRDNAKNTVYLQ NO: 41)MNNLKSEDTAVYYCALRAYS GRYYQFLEYDYWGQGTQVT VSS (SEQ ID NO: 4) DR930EVQLVESGGGLVQAGGSLRLS RTFSSYAVA ALSRGGGSA RNYDGTYYQECTVSGRTFSSYAVAWFRQAP (SEQ ID NO: 43) YYTDSVKG NQYNY GNVRELAAALSRGGGSAYYT(SEQ ID (SEQ ID NO: 45) DSVKGRFTISRDNAKNTVYLQ NO: 44)MNSLKPEDTAVYYCAARNYD GTYYQENQYNYWGQGTQVT VSS (SEQ ID NO: 5) DR931QVQLVESGGGLVQAGGSLLL RTFSTYAMG AISVNGGST RRPYPGSDFLTSCAASGRTFSTYAMGWFRQA (SEQ ID NO: 46) YYADSVTG WASYDYPGKERMFVAAISVNGGSTYY (SEQ ID (SEQ ID NO: 48) ADSVTGRFTISRDNAKNTMYLNO: 47) QMNNLKPGDTAVYYCAARRP YPGSDFLTWASYDYRGQGTL VTVSS (SEQ ID NO: 6)DR932 QVQLVESGGGLVQAGGFLRL RTFSYTTIG VISAGGGSR RRNTDTYTTTGSCAASGRTFSYTTIGWFRQAP (SEQ ID NO: 49) DYADALKG DYDYGKEREFVAVISAGGGSRDYAD (SEQ ID (SEQ ID NO: 51) ALKGRFTISRDNAKKMVYLQNO: 50) MNNLKPEDTAVYYCAVRRNT DTYTTTGDYDYWGQGTQVT VSS (SEQ ID NO: 7)DR933 QVQLVESGGGLVQPGDSLRLS GTISSLAMG AISWSGRST GEDGHSEYDYCVASGGTISSLAMGWFRQAP (SEQ ID NO: 52) YYVDSVKG (SEQ ID NO: 54)GKEREFVAAISWSGRSTYYVD (SEQ ID SVKGRFTISTDNAKNTVYLQM NO: 53)NSLKPEDTAVYYCVAGEDGH SEYDYWGQGTQVTVSS (SEQ ID NO: 8) DR934QVQLVESGGGLVQAGGSLRL RSFANYAMG AISRGGGST RSYSGSYTYSFSCAASGRSFANYAMGWFRQA (SEQ ID NO: 55) WYADSVKG GEYDYPGKERETVAAISRGGGSTWYA (SEQ ID (SEQ ID NO: 57) DSVKGRFTISKDNAKNTVYLQNO: 56) MNSLKPEDTAIYYCAARSYSG SYTYSFGEYDYWGQGTQVTV SS (SEQ ID NO: 9)DR935 QVQLVESGGGLVQAGGSLRL RAFSTYALG AISRGGGST RSYSSSYYYSQSCAASGRAFSTYALGWFRQA (SEQ ID NO: 58) DYADSVKG YEYDYPGKEREFIAAISRGGGSTDYA (SEQ ID (SEQ ID NO: 60) DSVKGRFTISRDNAKSTVYLQNO: 59) MNSLKPEDTAVYYCAARSYS SSYYYSQYEYDYWGQGTQVT VSS (SEQ ID NO: 10)DR936 EVQLVESGGGLVQAGGSLRLS RTFSSVAMA AISSGGGSTD RDYSSRRYYQSCAASGRTFSSVAMAWFRQAP (SEQ ID NO: 61) YADSVKG RYEYDLGKEREFVSAISSGGGSTDYAD (SEQ ID (SEQ ID NO: 63) SVKGRFTISKDNAKNTMYLQNO: 62) MDSLKPEDTAVYYCAARDYS SRRYYQSRYEYDLWGLGTQV TVSS (SEQ ID NO: 11)DR937 QVQLVESGGGLVQPGGSLRLS RTFRSYSMG AISWYSGTT NEIATMESSNDCAASGRTFRSYSMGWFRQAP (SEQ ID NO: 64) YYADPVKG Y GKEREFVAAISWYSGTTYYA(SEQ ID (SEQ ID NO: 66) DPVKGRFTISRDDAKNTLYLQ NO: 65)MNSLKPEDTAVYYCAANEIA TMESSNDYWGQGTQVTVSS (SEQ ID NO: 12) DR938QVQLVESGGGLVQPGGSLRLS RPRTTYAMG AISKAGGST RAGFAAQIFEYCTASGRPRTTYAMGWFRQAP (SEQ ID NO: 67) YVADSAKG DY GKEREIVAAISKAGGSTYVAD(SEQ ID (SEQ ID NO: 69) SAKGRFAISKDNAKNTVYLQ NO: 68)MNSLKPEDTAVYYCAARAGF AAQIFEYDYWGQGTLVTVSS (SEQ ID NO: 13) DR939EVQLVESGGGLVQAGGSMRL RTFGTYAMG SIDRDGSMS SRRAVISLQTVSCANSGRTFGTYAMGWFRQS (SEQ ID NO: 70) YYADSVKG DY PGKERERVASIDRDGSMSYYA(SEQ ID (SEQ ID NO: 72) DSVKGRFTISGDNAKNTVYLQ NO: 71)MNSLKPEDTAVYYCAASRRA VISLQTVDYWGQGTQVTVSS (SEQ ID NO: 14) DR940EVQLVESGGRLVQTGGSLRLS RTFSNYAMG AISWYSGNT NQIATMISVGDCAASGRTFSNYAMGWFRQAP (SEQ ID NO: 73) YYADSVKG Y GKEREFVAAISWYSGNTYYA(SEQ ID (SEQ ID NO: 75) DSVKGRFTISRDNAKNTMYL NO: 74)QMNSLKPEDTAVYYCAANQI ATMISVGDYWGQGTLVTVSS (SEQ ID NO: 15) DR941QVQLVESGGGLVEAGGSLRL SIFRLNLMG HVGTTGNTA DRWGQFS ACAASGSIFRLNLMGWYRQA(SEQ ID NO: 76) YADSVKG (SEQ ID NO: 78) PGKQRELVAHVGTTGNTAYA (SEQ IDDSVKGRFTISKDDAKNMVFL NO: 77) QMNSLKPEDTAVYYCYADR WGQFSWGQGTQVTVSS(SEQ ID NO: 16) DR942 QVQLVESGGGLVQAGGSLRL RTFTGYAMG VITWSGATTRIRDGVSPENP SCVASGRTFTGYAMGWFRQA (SEQ ID NO: 79) YYSASVKG NEYGYPGKEREFVAVITWSGATTYYS (SEQ ID (SEQ ID NO: 81) ASVKGRFTLSRDNAKNTVYLNO: 80) QMNSLKSEDTAVYYCAIRIRD GVSPENPNEYGYWGQGTQVT VSS (SEQ ID NO: 17)DR943 QVQLVESGGGLVQAGGSLRL RTVGYGMA AITWSGTST GSRRRVGVDVSCVASGRTVGYGMAWFRQAP (SEQ ID NO: 82) YYPDSVKG GGYDY GKQRDVVAAITWSGTSTYYP(SEQ ID (SEQ ID NO: 84) DSVKGRFTISRDNAKNTMYL NO: 83)QMSSLKPEDTAVYYCAAGSR RRVGVDVGGYDYWGQGTQV TVSS (SEQ ID NO: 18) DR944QVQLVESGGGLVQAGDSLRL RTFTFSTHNMG GIMWTSRAS AWYGNSGASYSCAASGRTFTFSTHNMGWFR (SEQ ID NO: 85) YADSVKG DY QAPGKEREFVGGIMWTSRAS(SEQ ID (SEQ ID NO: 87) YADSVKGRFTVSRDNAKNTV NO: 86)YLQMNSLKPEDTAVYYCAAA WYGNSGASYDYWGQGTQVT VSS (SEQ ID NO: 19) DR945QVQLVESGGGLVQLVQAGGS RTFSGYNMG AIAWAGSRT HDETYYRLDRVLRLSCAASGRTFSGYNMGWF (SEQ ID NO: 88) YYTDSVKG DLYTH RQAPGKEREFVAAIAWAGSR(SEQ ID (SEQ ID NO: 90) TYYTDSVKGRFTISRDNAKNT NO: 89)MYLQMNTLRPEDTAVYYCAA HDETYYRLDRVDLYTHWGQ GTQVTVSS (SEQ ID NO: 20) DR946QVQLVESGGGLVQPGESLRLS PFTRYAMG AISWSSGNT NEVATMSGPD CAASGPFTRYAMGWFRQAPG(SEQ ID NO: 91) YYVDSVKG DY KEREFVAAISWSSGNTYYVDS (SEQ ID(SEQ ID NO: 93) VKGRFTISRDNAKNTMYLQM NO: 92) NSLKPEDTAVYYCAANEVATMSGPDDYWGQGTQVTVSS (SEQ ID NO: 21) DR947 QVQLVESGGGLVQAGGSLRL GSFGRYTMGVISWSGTNT RETYYSHWDE SCAASGGSFGRYTMGWYRQA (SEQ ID NO: 94) YYADSVKGRMEYDY PGKEREFVAVISWSGTNTYYA (SEQ ID (SEQ ID NO: 96)DSVKGRFTISRDNAKNTMYL NO: 95) QMNDLKPEDTAVYYCAARET YYSHWDERMEYDYWGQGTQVTVSS (SEQ ID NO: 22) DR948 QVQLVESGGGLVQAGDSLRL SIDSSYYVS AINWGDSRTRIGLGGPVVAA SCVASGSIDSSYYVSWFRQAP (SEQ ID NO: 97) AYADSVKG PTRYPYGKERDLVAAINWGDSRTAYA (SEQ ID (SEQ ID NO: 99) DSVKGRFTISRDNAKNTVYLQNO: 98) MHSLRPNDTAVYYCASRIGLG GPVVAAPTRYPYWGQGTLVT VSS (SEQ ID NO: 23)DR949 QVQLVESGGGLVQAGGSLRL SILRFNVMS VITSGGSTN DESGQYYSCAASESILRFNVMSWLRQAP (SEQ ID NO: 100) YADSVKG (SEQ ID NO: 102)GKQRELVAVITSGGSTNYADS (SEQ ID VKGRFTISRDNAKNTVYLQM NO: 101)NSLKPEDTAVYYCAADESGQ YYWGQGTQVTVSS (SEQ ID NO: 24) DR950QVQLVESGGGLVQAGGSLRL LTTSSAALA TITSGGGSTY RFYSTTYYYRESCAASGLTTSSAALAWFRQAP (SEQ ID NO: 103) YADSVKG HEYSDGKERELDPTITSGGGSTYYAD (SEQ ID (SEQ ID NO: 105) SVKGRFTISKDNAKNTLYLQMNO: 104) SSLKPEDTAVYYCAARFYSTT YYYREHEYSDWGQGTQVTVS S (SEQ ID NO: 25)DR951 QVQLVESGGGLVQAGGSLRL RTFGTSFGSLS AISRNIGRTY TNSYDDLRRSYSCAASGRTFGTSFGSLSMGWF MG YADSVKD AYDY RQAPGKEREFVAAISRNIGRT(SEQ ID NO: 106) (SEQ ID (SEQ ID NO: 108) YYADSVKDRFTISRDNAKNTA NO: 107)SLQMNSLEPEDTAVYNCAAT NSYDDLRRSYAYDYWGQGT QVTVSS (SEQ ID NO: 26) DR952QVQLVESGGGLVQAGGFLRL RTFSSLAMA AISRSGGSTD RDYSTLQYYNESCAASGRTFSSLAMAWFRQA (SEQ ID NO: 109) YADSVKG YEYSDPGKEREFVAAISRSGGSTDYA (SEQ ID (SEQ ID NO: 111) DSVKGRFTISRDNAKSTLYLQNO: 110) MSSLKPEDTAVYYCAARDYS TLQYYNEYEYSDWGQGTLVT VSS (SEQ ID NO: 27)DR953 QVQLVESGGGLVQAGDSLRL PTFSTYAMA AITQSGRTT RDLWSDSPDDSCAASGPTFSTYAMAWFRQA (SEQ ID NO: 112) YYEDSVKG WRIYSFPGKEREFVAAITQSGRTTYYE (SEQ ID (SEQ ID NO: 114) DSVKGRFTISKDNAKNTLYLQNO: 113) MNSLQPEDTAVYYCAARDLW SDSPDDWRIYSFWGQGTQVT VSS (SEQ ID NO: 28)DR954 QVQLVESGGGLVQAGGSLRL RTLHNFAMA AISKGGGSA NDLASYSDSSYSCAAARRTLHNFAMAWFRQA (SEQ ID NO: 115) DYADSVKG TSTSRYDYPGKEREFVAAISKGGGSADYA (SEQ ID (SEQ ID NO: 117) DSVKGRFTISRDNAKNTVYLQNO: 116) MNSLKPEDTAVYYCAANDLA SYSDSSYTSTSRYDYWGQGT QVTVSS(SEQ ID NO: 29) DR955 QVQLVESGGGLVQTGGSLRLS RTFSSYAMA AISILGGSADRRPAPSDSYWS CAASGRTFSSYAMAWFRQAP (SEQ ID NO: 118) YEDSVQG STSYAYGKEREFVAAISILGGSADYEDS (SEQ ID (SEQ ID NO: 120) VQGRFTISRDNAKNTMYLQMNO: 119) NSLKPEDTAVYYCAARRPAPS DSYWSSTSYAYWGQGTLVTV SS (SEQ ID NO: 30)DR956 EVQLVESGGGLVQAGGSLRLS RTFSSLAMA ATTILGGSA RRPAPSDNYWSCAASGRTFSSLAMAWFRQAP (SEQ ID NO: 121) DYGDPVKG PASYAYGKEREFVAATTILGGSADYGD (SEQ ID (SEQ ID NO: 123) PVKGRFTISRDNAKNTVYLQNO: 122) MNSLKPEDTAVYYCTGRRPA PSDNYWSPASYAYWGQGTQV TVSS (SEQ ID NO: 31)DR957 QVQLVESGGGLVQAGGSLRL RTFSSYAMA AITVSGAST GGPGTIFPDYDSCAPSGRTFSSYAMAWFRQPP (SEQ ID NO: 124) YYADSVKG Y GKEREFVAAITVSGASTYYAD(SEQ ID (SEQ ID NO: 126) SVKGRFTISRDNAKNSMYLQ NO: 125)MNSLKPEDTAVYYCAAGGPG TIFPDYDYWGQGTQVTVSS (SEQ ID NO: 32) DR958QVQLVESGGGLVQAGDSLTL RTFSGYNMG AINWIGGAT YSEKFYSGKDYSCTASGRTFSGYNMGWFRQA (SEQ ID NO: 127) YYADSVKG YTRDYDYPGKERDFVAAINWIGGATYY (SEQ ID (SEQ ID NO: 129) ADSVKGRFTISRDNAKNTVYLNO: 128) QMNSLKPEDTAVYYCHRYSE KFYSGKDYYTRDYDYWGQG TQVTVSS(SEQ ID NO: 33)

In some embodiments, the foregoing sets of CDRs are incorporated in ahumanized VHH framework to provide “humanized” sdAb IFNgR2 bindingmolecules.

The disclosure further provides methods of chemical or recombinantprocesses for the preparation of the IFNgR2 binding molecules of thepresent disclosure.

The disclosure further provides nucleic acids encoding the IFNgR2binding molecules. Table 2 below provides examples of DNA sequencesencoding IFNgR2 binding molecules as described herein.

TABLE 2 DNA Sequences Encoding IFNgR2 VHHs NAME DNA Sequence DR927CAGGTGCAGCTGGTGGAGAGCGGAGGCGGACTGGTACAAGCAGGGGGTTCTCTCAGGCTGAGCTGTGCGGCTTCCGGGAGGACTTTCACCTCTTATGCTATGAATTGGTTCCGCCAGGCCCCTGGGAAGGAAAGGGAGTTTGTGGCAGCCATCTCTTGGTCCAGCGGCAATACCTACGTGGCCGACTCCGTCAAGGGTAGGTTCGCCATCAGCCGGGACAAAGCTAAGAATACTATGTACTTGCAGATGAACAGCCTCGCGCCGGAAGATACTGCGGTGTATTACTGCGCCGCTACTACCATCGCCACGATGTCCGACGAGTATACATATTGGGGACAGGGAACTCAGGTTACAGTATCCTCC (SEQ ID NO: 130) DR928CAGGTCCAGCTGGTCGAATCCGGTGGCGGGTTGGTACAGGCGGGTGGCTCCCTGCGCCTGAGCTGCGCCGCGAGTGGGCGTACATTTTCTAACTACAGAATGGGCTGGTTCAGGCAGGCTCCGGGAAAGGAGCGTGAGTTCGTGGCTGCCATTTCACGCGGAGGTGGCACCACACTGTACGCCGACTCTGTCAAAGGCCGCTTCACCATCTCTCGCGACAACGCTAAGAATACTGTTGATCTCCAGATGAACCGCCTGAAGCCGGAAGACACCGCAGTTTACTTTTGTGCCGCAGGCGATTTCTCTACCACTTGGGACGAGTATAACTACTGGGGGCAGGGAACACAAGTGACCGTGTCCAGT (SEQ ID NO: 131) DR929CAGGTTCAACTGGTGGAGAGCGGTGGGGGCCTGGTGCAGGCAGGCGGGAGCTTGCGGCTTTCATGTGTTGCATCTGGCCGCACCTTCTCAAGCTATGCGATGGGCTGGTTCCGTCAGACTCCGGGGAAAGAGAGGGAGTTCGTGAGCGCGATTTCTCGCGGTGGAGGCAGCACCGACTATGCCGATTCCGTGAAGGGCAGGTTTACGATCTCCAGAGATAACGCCAAGAACACAGTTTATTTGCAGATGAATAACCTGAAATCCGAGGACACCGCAGTGTATTACTGCGCCCTGCGGGCCTATTCAGGCCGCTATTACCAGTTCCTGGAGTACGATTACTGGGGCCAGGGCACACAGGTGACTGTGTCCTC C (SEQ ID NO: 132) DR930GAGGTTCAGTTGGTGGAGTCTGGGGGCGGTCTGGTCCAGGCTGGTGGATCATTGCGCCTGAGCTGTACCGTTTCAGGCAGGACTTTTTCTAGCTACGCCGTAGCTTGGTTCCGCCAGGCACCTGGCAACGTCCGGGAGCTGGCGGCTGCCCTGAGTCGCGGGGGAGGCTCTGCTTACTATACAGACAGTGTCAAGGGTCGCTTCACTATTAGCCGCGACAATGCGAAAAACACCGTCTACCTTCAGATGAACAGTCTGAAGCCCGAAGACACTGCGGTGTATTACTGCGCGGCCAGGAACTACGACGGCACCTATTACCAGGAAAACCAATACAATTACTGGGGGCAGGGAACCCAGGTGACCGTCAGCA GC (SEQ ID NO: 133) DR931CAGGTCCAGCTGGTGGAATCAGGCGGAGGCTTGGTGCAGGCCGGGGGCAGTCTGTTGCTGTCTTGCGCCGCGAGCGGACGCACATTCTCCACCTACGCTATGGGGTGGTTCCGCCAGGCACCTGGAAAAGAACGCATGTTTGTCGCGGCCATCTCCGTTAACGGAGGCAGTACCTACTATGCAGATTCTGTTACGGGCCGTTTCACCATTAGCCGCGACAATGCGAAAAACACCATGTATTTGCAAATGAATAACCTGAAGCCTGGTGACACAGCCGTGTATTACTGCGCAGCGCGTCGCCCCTACCCCGGTTCCGACTTTCTCACATGGGCCTCCTACGATTACAGGGGCCAGGGCACCCTGGTGACCGTGTC TAGT (SEQ ID NO: 134)DR932 CAGGTTCAGCTGGTCGAATCTGGCGGTGGACTGGTGCAAGCTGGTGGGTTCCTGCGCCTCAGCTGTGCCGCTAGTGGCCGTACCTTTAGCTATACAACCATCGGCTGGTTCCGCCAGGCTCCAGGGAAGGAACGCGAGTTCGTCGCCGTGATCTCAGCAGGAGGCGGTTCCCGCGATTACGCGGACGCCCTCAAAGGACGCTTTACAATCTCTCGCGATAACGCTAAAAAGATGGTTTATTTGCAAATGAATAACTTGAAGCCCGAGGATACCGCCGTGTATTACTGCGCCGTGAGGCGGAATACTGACACATATACCACAACCGGCGACTACGACTACTGGGGTCAGGGCACCCAGGTTACCGTTTCATCC (SEQ ID NO: 135) DR933CAAGTTCAGCTTGTAGAGTCTGGCGGGGGCCTGGTGCAACCCGGTGACTCACTGAGGCTGTCTTGTGTGGCCTCCGGGGGTACAATTTCCTCACTGGCTATGGGTTGGTTCAGGCAGGCTCCGGGTAAGGAGAGGGAGTTTGTCGCAGCCATCAGCTGGTCTGGCCGCTCAACATATTACGTGGATAGCGTGAAAGGCCGCTTCACTATTTCTACTGATAACGCGAAGAATACCGTGTATCTCCAAATGAACTCCCTGAAACCGGAGGACACAGCGGTGTACTATTGCGTCGCAGGCGAGGATGGACACAGCGAGTATGACTATTGGGGCCAAGGCACCCAGGTCACAGTGTCCTCA (SEQ ID NO: 136) DR934CAGGTGCAGCTTGTGGAGAGCGGAGGCGGGCTGGTGCAGGCCGGGGGTAGCTTGCGCCTCAGCTGTGCTGCCTCTGGACGCTCATTTGCCAACTACGCAATGGGCTGGTTCCGTCAGGCTCCTGGGAAAGAGCGCGAGACCGTGGCGGCCATCAGTCGCGGGGGAGGCAGCACGTGGTACGCGGACTCAGTCAAGGGGAGGTTTACTATTTCCAAGGATAACGCTAAGAACACCGTGTATCTGCAAATGAACAGCCTCAAGCCCGAGGACACAGCAATCTACTATTGCGCGGCCCGCAGTTACTCCGGCTCCTACACTTATTCCTTCGGCGAGTACGACTACTGGGGTCAGGGCACCCAAGTTACCGTGTC CTCC (SEQ ID NO: 137)DR935 CAGGTGCAGCTGGTTGAGTCTGGAGGCGGTCTTGTCCAGGCTGGTGGCTCCTTGCGCCTGAGCTGTGCCGCTTCCGGGAGAGCTTTCTCCACATACGCTCTCGGCTGGTTCAGGCAGGCCCCCGGAAAGGAGCGCGAGTTCATCGCTGCAATCAGCCGGGGCGGTGGCAGCACTGATTATGCAGATTCTGTGAAGGGACGCTTCACCATCTCTCGTGATAACGCCAAGTCTACCGTATATTTGCAGATGAATAGTCTGAAGCCCGAAGACACCGCTGTGTATTACTGTGCAGCGCGTAGCTACTCCTCTAGTTACTATTACTCTCAGTACGAGTACGACTACTGGGGACAGGGTACTCAGGTGACTGTGTCCAG C (SEQ ID NO: 138) DR936GAGGTCCAGCTCGTGGAGAGTGGCGGTGGCCTCGTGCAGGCCGGGGGCTCCCTGAGACTGAGTTGTGCCGCTTCTGGCCGCACCTTCAGTTCAGTCGCTATGGCTTGGTTTCGGCAGGCCCCTGGCAAGGAGCGCGAGTTCGTGTCCGCTATCAGTTCTGGAGGCGGTTCCACGGATTACGCTGATAGCGTGAAGGGCAGATTCACTATCAGCAAGGACAACGCTAAGAACACCATGTATCTCCAGATGGACTCTCTTAAACCCGAGGATACCGCCGTGTATTACTGCGCCGCTCGCGACTATTCTAGCCGTCGCTATTACCAGTCCCGGTACGAGTACGACCTCTGGGGCCTGGGAACCCAAGTCACAGTCT CCTCC (SEQ ID NO: 139)DR937 CAGGTGCAGTTGGTGGAGTCCGGTGGGGGCCTGGTGCAGCCAGGTGGAAGCCTCCGCCTGTCCTGTGCTGCAAGTGGCCGCACCTTTAGGTCCTACTCAATGGGCTGGTTTCGTCAGGCTCCAGGAAAGGAACGGGAGTTCGTTGCCGCTATTTCCTGGTATTCCGGGACAACGTACTATGCCGACCCAGTCAAGGGTAGATTCACTATCAGCCGGGACGATGCAAAGAACACTCTCTACTTGCAGATGAACTCCCTGAAGCCCGAGGACACCGCCGTGTATTACTGCGCGGCTAACGAGATCGCCACAATGGAATCCTCTAACGATTACTGGGGCCAGGGGACCCAGGTTACCGTCTCTAGC (SEQ ID NO: 140) DR938CAGGTGCAGCTGGTCGAGAGTGGAGGGGGATTGGTCCAGCCTGGTGGCTCTCTGCGCCTGTCATGCACTGCTTCAGGCCGCCCCCGTACAACGTATGCAATGGGCTGGTTCCGTCAGGCTCCCGGCAAGGAGCGCGAGATCGTGGCGGCTATTTCCAAAGCCGGAGGCTCTACCTACGTGGCCGACTCTGCGAAAGGCCGCTTTGCCATCTCTAAGGACAACGCCAAGAATACCGTCTATCTGCAAATGAACAGCCTCAAGCCTGAGGATACCGCAGTTTATTACTGTGCAGCTCGCGCAGGCTTCGCTGCCCAAATCTTCGAGTACGACTACTGGGGACAGGGCACCCTGGTGACAGTGTCTAGC (SEQ ID NO: 141) DR939GAGGTTCAACTGGTCGAGTCCGGGGGCGGGCTCGTGCAGGCCGGAGGTTCCATGCGCCTGTCCTGTGCCAATTCCGGTAGAACCTTTGGGACCTATGCTATGGGCTGGTTCCGCCAGTCACCCGGCAAAGAGCGCGAGCGGGTCGCGTCCATCGACCGTGACGGTTCTATGTCCTATTACGCCGACAGCGTGAAAGGCAGGTTCACAATCAGCGGAGACAATGCGAAGAACACCGTGTATCTCCAGATGAACTCCCTGAAACCTGAGGACACCGCCGTCTATTACTGCGCGGCTTCAAGACGCGCCGTTATTTCTCTCCAGACAGTTGATTACTGGGGCCAGGGAACCCAGGTGACTGTGTCCAGC (SEQ ID NO: 142) DR940GAAGTGCAGCTCGTGGAGTCTGGTGGCCGTTTGGTCCAGACCGGAGGCAGCTTGAGGCTCTCATGCGCTGCCTCTGGCAGGACATTTTCCAATTATGCAATGGGATGGTTTCGTCAGGCTCCTGGCAAGGAGAGGGAGTTCGTGGCTGCAATCTCTTGGTACAGCGGCAACACATACTATGCCGACTCCGTGAAAGGCAGGTTTACCATCAGCCGGGATAACGCCAAAAACACTATGTATCTCCAGATGAACTCCCTGAAGCCAGAGGATACGGCGGTCTATTACTGTGCAGCCAATCAAATCGCCACTATGATTAGCGTGGGCGACTACTGGGGCCAAGGCACCCTGGTGACAGTGTCATCC (SEQ ID NO: 143) DR941CAGGTGCAGTTGGTCGAGTCAGGAGGGGGCCTGGTCGAGGCCGGGGGCTCCCTGAGGCTCGCTTGCGCTGCGAGTGGATCAATCTTCCGTTTGAACCTGATGGGATGGTATAGACAGGCTCCTGGCAAGCAGCGTGAACTGGTCGCACACGTGGGTACTACAGGAAACACCGCCTACGCCGACTCTGTCAAGGGGCGCTTTACCATCTCAAAGGACGATGCTAAGAACATGGTGTTTCTCCAGATGAACTCCCTCAAGCCCGAGGATACTGCCGTCTATTACTGCTATGCGGACCGCTGGGGTCAGTTCTCCTGGGGCCAGGGAA CACAGGTAACCGTTTCTTCA(SEQ ID NO: 144) DR942 CAGGTGCAGCTCGTGGAGTCTGGCGGTGGCCTGGTCCAGGCCGGAGGCAGCCTCCGGTTGAGCTGTGTAGCTAGTGGCCGTACCTTTACGGGCTACGCGATGGGCTGGTTCAGACAAGCGCCTGGCAAGGAGAGGGAGTTCGTCGCCGTGATTACTTGGTCCGGGGCCACTACCTATTACTCCGCTTCCGTGAAGGGCCGTTTCACGCTGAGCCGGGATAACGCCAAAAACACAGTGTACCTCCAGATGAACTCCCTGAAGTCCGAGGACACTGCCGTTTATTACTGCGCCATTCGCATCCGGGACGGAGTGTCTCCTGAAAACCCTAACGAGTACGGTTACTGGGGCCAGGGCACCCAGGTAACCGTGTCCT CA (SEQ ID NO: 145) DR943CAGGTCCAGCTCGTGGAGTCCGGCGGGGGCTTGGTTCAGGCTGGGGGAAGCCTGCGTCTGTCATGCGTGGCGAGCGGGAGGACCGTCGGCTACGGCATGGCTTGGTTTAGGCAGGCTCCTGGCAAGCAACGCGATGTTGTGGCCGCGATCACTTGGTCCGGCACTTCCACCTATTACCCCGACTCAGTCAAGGGGCGCTTCACCATCTCCAGAGATAACGCCAAGAACACTATGTATCTCCAGATGTCATCTCTGAAGCCAGAAGATACCGCTGTATATTACTGCGCCGCTGGCTCCCGTCGCCGGGTGGGCGTGGACGTGGGTGGATACGACTACTGGGGCCAAGGCACTCAGGTGACCGTCTCCAGC (SEQ ID NO: 146) DR944CAGGTGCAGCTCGTCGAGAGCGGGGGCGGACTGGTACAGGCTGGCGACTCCTTGCGTCTGAGCTGCGCCGCGTCCGGCAGGACGTTCACCTTCTCAACTCACAATATGGGCTGGTTCCGTCAGGCACCGGGTAAGGAGAGAGAGTTCGTCGGAGGCATCATGTGGACCAGCCGGGCAAGTTACGCCGATTCTGTGAAGGGTCGTTTCACCGTAAGCCGTGATAACGCGAAGAACACTGTCTACTTGCAGATGAACTCTCTGAAGCCCGAGGACACTGCGGTTTACTATTGCGCTGCCGCATGGTATGGTAACAGCGGAGCCTCCTACGACTACTGGGGTCAAGGAACTCAGGTCACGGTCAGCTCC (SEQ ID NO: 147) DR945CAGGTCCAACTGGTCGAGTCTGGCGGAGGCCTCGTGCAACTTGTTCAAGCCGGGGGCAGCCTGCGCCTTTCTTGTGCTGCGAGTGGCCGCACCTTTTCCGGGTATAACATGGGTTGGTTTCGTCAGGCCCCAGGTAAGGAGCGCGAGTTCGTGGCTGCCATTGCTTGGGCAGGCAGCAGGACATATTACACCGATAGCGTCAAGGGGCGGTTTACTATCTCTCGCGACAATGCAAAAAACACTATGTACCTCCAGATGAACACCCTGCGTCCTGAGGATACAGCCGTGTATTACTGCGCAGCCCATGACGAGACCTATTACCGCCTTGACCGGGTGGACCTCTATACCCACTGGGGCCAGGGAACCCAGGTCAC TGTGTCCTCC(SEQ ID NO: 148) DR946 CAGGTGCAACTGGTTGAGTCCGGGGGCGGACTGGTGCAACCTGGCGAGAGTCTGCGCCTGTCCTGCGCGGCCTCCGGTCCTTTTACTAGATATGCGATGGGTTGGTTCCGTCAAGCGCCTGGGAAGGAGCGTGAGTTCGTCGCGGCTATCAGCTGGTCATCTGGAAATACGTATTACGTGGATTCCGTGAAGGGCAGGTTCACTATTTCCAGAGACAACGCTAAGAACACAATGTACTTGCAGATGAACAGCCTGAAGCCAGAGGACACAGCGGTTTACTATTGTGCTGCAAACGAGGTCGCCACCATGAGTGGACCCGATGACTATTGGGGACAAGGTACACAAGTCACTGTGTCCTCT (SEQ ID NO: 149) DR947CAGGTGCAGCTGGTGGAGTCTGGAGGTGGGCTGGTGCAGGCGGGAGGCTCTCTGCGTCTGTCTTGTGCTGCCAGCGGCGGTTCCTTTGGCCGGTACACAATGGGATGGTATCGTCAAGCGCCTGGTAAGGAGCGCGAGTTTGTTGCTGTGATCTCATGGAGCGGCACAAACACGTACTATGCGGACAGCGTGAAGGGTCGCTTTACCATTTCCCGTGATAACGCGAAGAATACCATGTACCTGCAAATGAACGATTTGAAACCCGAAGATACCGCAGTTTATTACTGTGCTGCGCGTGAGACTTATTACTCACACTGGGACGAAAGGATGGAGTATGACTATTGGGGCCAGGGCACCCAGGTTACAGTATCTTC C (SEQ ID NO: 150) DR948CAGGTCCAGCTCGTAGAAAGCGGAGGTGGCCTTGTCCAAGCTGGCGACAGCCTGCGCCTTTCCTGCGTTGCCAGCGGCTCCATCGACTCATCCTATTACGTGTCTTGGTTCCGTCAGGCACCAGGCAAGGAACGTGATCTGGTAGCTGCCATCAACTGGGGCGATTCTCGCACCGCCTATGCTGACTCCGTTAAGGGCCGGTTCACGATCTCCCGTGATAACGCGAAAAATACTGTGTATCTCCAGATGCACTCCCTGCGCCCCAACGATACCGCAGTGTATTACTGTGCCAGCAGAATTGGGCTGGGCGGGCCGGTGGTTGCGGCCCCCACCCGTTATCCCTATTGGGGCCAGGGCACCCTGGTAACCGTGTC TTCC (SEQ ID NO: 151)DR949 CAAGTGCAACTGGTAGAGTCAGGCGGTGGCCTTGTGCAGGCCGGTGGCTCTCTGCGCCTGTCTTGCGCTGCCTCTGAGTCTATCTTGCGGTTTAACGTGATGTCATGGTTGCGTCAGGCACCAGGGAAACAGCGTGAGCTGGTGGCAGTCATCACTAGCGGTGGCAGCACGAATTATGCAGACTCCGTGAAAGGCCGCTTCACGATCTCTCGTGACAACGCTAAAAACACCGTGTATCTCCAGATGAACAGCCTGAAGCCAGAAGACACTGCCGTGTATTACTGTGCTGCCGACGAGAGCGGGCAGTATTACTGGGGACAGGGC ACTCAGGTGACAGTCAGCAGC(SEQ ID NO: 152) DR950 CAGGTCCAGCTCGTGGAGTCTGGGGGCGGTCTCGTCCAGGCTGGGGGTTCCCTGCGGCTGAGCTGTGCCGCTTCTGGCTTGACGACCAGTAGCGCCGCTCTGGCCTGGTTTCGTCAGGCTCCTGGCAAGGAGCGTGAGCTTGATCCAACTATTACCAGCGGCGGAGGCTCTACCTACTATGCCGACAGCGTTAAGGGTCGGTTTACCATCAGCAAAGACAACGCTAAGAACACTCTTTATCTCCAGATGTCAAGCCTCAAACCTGAGGACACCGCCGTCTACTATTGCGCCGCTCGCTTTTATAGCACCACTTACTATTACCGCGAACACGAGTATAGTGACTGGGGGCAGGGCACCCAAGTCACAGTGAGCA GC (SEQ ID NO: 153) DR951CAGGTGCAACTGGTGGAATCTGGGGGTGGCTTGGTGCAGGCTGGCGGGTCTCTGCGTCTGTCCTGTGCGGCTTCAGGACGCACTTTCGGCACCAGCTTTGGCAGCCTGAGCATGGGCTGGTTCAGACAAGCCCCCGGCAAAGAGCGTGAGTTCGTGGCTGCCATCTCTCGCAACATTGGCCGTACTTATTACGCAGATTCCGTGAAAGACAGGTTTACGATCTCTCGCGACAACGCTAAGAATACCGCCTCTCTTCAGATGAACTCTCTGGAGCCCGAAGACACTGCTGTCTATAATTGTGCCGCGACAAACTCCTACGATGACCTTAGGCGCTCCTACGCCTACGACTACTGGGGACAGGGCACCCAGGTGA CAGTCAGCTCC(SEQ ID NO: 154) DR952 CAGGTCCAGCTGGTGGAGAGCGGCGGTGGGCTGGTTCAGGCTGGAGGCTTCCTGCGCCTGTCTTGCGCTGCCTCAGGTCGTACTTTTTCCTCTTTGGCAATGGCTTGGTTCCGTCAAGCTCCCGGCAAGGAGCGCGAGTTCGTAGCCGCTATCTCCCGCTCTGGAGGCTCTACCGACTACGCTGACAGCGTCAAGGGACGCTTCACCATCTCTCGGGACAACGCCAAGTCCACCCTGTACTTGCAGATGTCTAGCCTGAAACCTGAGGATACAGCGGTGTATTACTGCGCTGCACGTGACTACTCTACTCTCCAGTATTACAATGAGTATGAATACTCCGACTGGGGCCAAGGCACTCTGGTCACTGTGTCCTCA (SEQ ID NO: 155) DR953CAGGTGCAGCTCGTAGAGTCTGGCGGTGGCCTGGTCCAGGCTGGGGACTCACTGCGTCTGAGTTGTGCCGCGTCCGGCCCTACCTTCAGTACGTATGCAATGGCCTGGTTTCGCCAAGCTCCAGGTAAGGAACGCGAGTTCGTGGCTGCCATTACCCAGAGTGGTCGCACCACTTACTATGAAGATTCTGTGAAGGGGCGGTTCACCATTAGCAAGGATAATGCCAAGAACACCCTCTATCTCCAGATGAACAGCCTCCAGCCTGAAGATACGGCGGTGTATTACTGCGCGGCCCGCGACTTGTGGTCTGATTCTCCCGATGACTGGAGAATTTACTCTTTCTGGGGACAGGGGACCCAAGTAACAGTCAGTTC C (SEQ ID NO: 156) DR954CAGGTTCAGCTTGTGGAGTCCGGTGGCGGTCTGGTCCAGGCAGGTGGCTCCCTGCGGCTGTCTTGCGCAGCCGCTCGTCGCACACTGCACAACTTCGCAATGGCGTGGTTTCGCCAGGCCCCTGGTAAGGAGAGGGAGTTTGTGGCTGCCATTTCTAAGGGAGGCGGTTCCGCCGACTACGCCGATAGCGTTAAGGGGCGCTTTACCATTAGTAGGGACAACGCTAAGAACACAGTGTACCTCCAGATGAACAGCCTCAAGCCAGAGGATACCGCAGTTTACTATTGCGCTGCCAACGATCTGGCTTCTTATAGCGACAGCTCTTACACATCCACAAGCCGCTACGACTACTGGGGTCAGGGCACCCAGGT TACAGTGTCCTCT(SEQ ID NO: 157) DR955 CAGGTGCAGCTCGTCGAGTCTGGCGGGGGACTGGTGCAGACTGGTGGCTCCCTGCGCCTCTCTTGCGCGGCCTCTGGCCGCACCTTCTCATCCTATGCTATGGCTTGGTTCCGTCAAGCGCCCGGTAAAGAGAGGGAGTTTGTGGCCGCTATCTCTATCCTCGGTGGCAGCGCTGACTACGAGGATTCCGTTCAGGGACGCTTCACTATCTCAAGAGATAACGCCAAGAACACTATGTACCTTCAGATGAACTCCCTCAAGCCAGAGGACACCGCTGTGTATTACTGTGCCGCTCGCAGACCGGCTCCCTCCGACAGCTACTGGAGCAGTACAAGTTACGCATACTGGGGACAGGGGACCCTGGTGACAGTGTC CAGT (SEQ ID NO: 158)DR956 GAGGTACAGCTGGTCGAATCCGGTGGCGGTCTGGTGCAGGCTGGCGGAAGCCTGCGCTTGAGCTGTGCGGCCTCTGGCCGCACTTTCAGCTCTCTTGCAATGGCGTGGTTCCGCCAGGCCCCTGGAAAGGAGCGCGAGTTCGTGGCCGCGACCACAATCCTCGGAGGCTCCGCCGACTACGGCGACCCGGTCAAGGGCCGCTTCACAATCTCCCGCGATAATGCTAAGAACACGGTGTATCTCCAGATGAACTCACTGAAGCCAGAGGATACCGCCGTTTATTACTGCACTGGGAGACGCCCCGCTCCATCCGACAACTACTGGTCCCCTGCCAGCTATGCTTACTGGGGACAAGGCACCCAAGTTACCGT GAGCAGT (SEQ ID NO: 159)DR957 CAGGTGCAGTTGGTGGAAAGCGGCGGTGGCCTTGTGCAGGCTGGCGGTTCACTGCGCCTCAGCTGCGCACCTTCCGGTCGCACCTTCTCCTCTTACGCGATGGCGTGGTTTCGCCAGCCTCCAGGCAAGGAACGGGAGTTTGTGGCTGCCATTACCGTTTCCGGCGCAAGTACCTATTACGCTGACTCTGTTAAGGGCCGTTTCACCATCTCTCGCGATAATGCGAAAAACTCTATGTACCTTCAGATGAACTCTTTGAAACCGGAGGATACCGCTGTGTACTATTGTGCAGCTGGCGGTCCCGGAACCATCTTCCCAGACTATGATTACTGGGGTCAGGGTACTCAGGTCACCGTCTCCTCC (SEQ ID NO: 160) DR958CAAGTACAGCTCGTCGAATCCGGCGGTGGACTGGTGCAGGCCGGAGATTCCCTTACCCTGTCTTGCACCGCTTCCGGTCGCACCTTTTCAGGTTACAATATGGGCTGGTTCAGGCAGGCCCCTGGTAAGGAGCGCGACTTCGTCGCTGCCATCAACTGGATCGGAGGTGCTACCTATTACGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCAGGGATAATGCCAAGAACACTGTCTACCTGCAAATGAACAGCCTGAAGCCCGAGGATACTGCCGTGTATTACTGCCATCGCTATTCCGAGAAGTTCTACAGCGGGAAGGACTACTATACTCGTGACTACGACTACTGGGGTCAGGGTACACAGGTAACTGT GTCCTCA (SEQ ID NO: 161)

The disclosure further provides recombinant viral and non-viral vectorscomprising a nucleic acid encoding the IFNgR2 binding molecules of thepresent disclosure or the CDRs of the IFNgR2 binding molecules of thepresent disclosure.

The disclosure further provides host cells comprising recombinant viraland non-viral vectors comprising a nucleic acid the IFNgR2 bindingmolecules of the present disclosure or the CDRs of the IFNgR2 bindingmolecules of the present disclosure.

The disclosure further provides host cells comprising recombinant viraland non-viral vectors comprising a nucleic acid the IFNgR2 bindingmolecules of the present disclosure or the CDRs of the IFNgR2 bindingmolecules of the present disclosure.

The disclosure further provides pharmaceutical formulations comprisingthe recombinant viral and non-viral vectors comprising a nucleic acidthe IFNgR2 binding molecules of the present disclosure and methods ofuse thereof in the treatment or prevention of diseases, disorders orconditions in a mammalian subject.

The disclosure further provides kits comprising the IFNgR2 bindingmolecules of the present disclosure.

In another aspect, the present disclosure provides constructs for thetargeted delivery of therapeutic agents to a cell expressing the IFNgR2receptor, wherein the IFNgR2 binding molecule is conjugated to one ormore therapeutic agents, optionally through a chemical or polypeptidelinker.

In some embodiments, the IFNgR2 binding molecules of the presentdisclosure are useful to inhibit the activity of interferon gamma invitro and/or in vivo. In some embodiments, the IFNgR2 binding moleculesof the present disclosure are useful in the treatment of autoimmunediseases. The disclosure further provides methods of use of theforegoing in the treatment of an autoimmune disease in a subject, themethod comprising administering to the subject a therapeuticallyeffective amount IFNgR2 binding molecule of the present disclosure. Insome embodiments, the IFNgR2 binding molecules of the present disclosuremay be used alone or in combination with one or more supplementarytherapeutic agents. In some embodiments, the diseases amenable totreatment are diseases, disorders or conditions associated withsignaling from receptor comprising the IFNgR2. In some embodiments, theIFNgR2 binding molecules of the present disclosure are useful in thetreatment of diseases associated with dysregulated T cell or B cellactivity. In some embodiments, the IFNgR2 binding molecules of thepresent disclosure are useful in the treatment of autoimmune diseases.In some embodiments, the IFNgR2 binding molecule of the presentdisclosure is administered to a subject in a pharmaceutically acceptableformulation. In some embodiments, the IFNgR2 binding molecule of thepresent disclosure is administered by the administration to the subjectof a composition comprising a recombinant viral or non-viral vectorcomprising a nucleic acid sequence encoding the IFNgR2 binding moleculeof the present disclosure.

In another aspect, the present disclosure provides constructs for theidentification of cells expressing the IFNgR2 receptor wherein theIFNgR2 binding molecule is conjugated to one or more imaging agents,optionally through a chemical or polypeptide linker. The disclosurefurther provides methods of use of the foregoing in the identificationof cells expressing the IFNgR2 receptor in a subject, the methodcomprising the administration of an effective amount of the IFNgR2binding molecule conjugated to the imaging agent to a subject in need oftreatment and evaluating the subject for the presence of the imagingagent that is conjugated to the IFNgR2 binding molecule.

In another aspect, the present disclosure provides IFNgR2 bindingmolecules which have been modified for extended duration of action invivo wherein the IFNgR2 binding molecule is conjugated to one or morecarrier molecules.

The present disclosure provides IFNgR2 binding molecules comprising apolypeptide sequence that specifically binds to the extracellular domainof the IFNgR2 and methods of use thereof in the isolation, depletion orenrichment of cells expressing the IFNgR2 receptor in a biologicalsample.

DETAILED DESCRIPTION OF THE INVENTION Introduction

In order for the present disclosure to be more readily understood,certain terms and phrases are defined below as well as throughout thespecification. The definitions provided herein are non-limiting andshould be read in view of the knowledge of one of skill in the art wouldknow.

Before the present methods and compositions are described, it is to beunderstood that this disclosure is not limited to particular method orcomposition described, as such may, of course, vary.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

It should be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof,e.g., polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

It will be appreciated that throughout this disclosure reference is madeto amino acids according to the single letter or three letter codes. Forthe reader's convenience, the single and three letter amino acid codesare provided in Table 3 below:

TABLE 3 Amino Acid Abbreviations Single Letter 3-letter AbbreviationName abbreviation G Glycine Gly P Proline Pro A Alanine Ala V Valine ValL Leucine Leu I Isoleucine Ile M Methionine Met C Cysteine Cys FPhenylalanine Phe Y Tyrosine Tyr W Tryptophan Trp H Histidine His KLysine Lys R Arginine Arg Q Glutamine Gln N Asparagine Asn EGlutamic Acid Glu D Aspartic Acid Asp S Serine Ser T Threonine Thr

Standard methods in molecular biology are described in the scientificliterature (see, e.g., Sambrook and Russell (2001) Molecular Cloning,3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloningin bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammaliancells and yeast (Vol. 2), glycoconjugates and protein expression (Vol.3), and bioinformatics (Vol. 4)). The scientific literature describesmethods for protein purification, including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization, aswell as chemical analysis, chemical modification, post-translationalmodification, production of fusion proteins, and glycosylation ofproteins (see, e.g., Coligan, et al. (2000) Current Protocols in ProteinScience, Vols. 1-2, John Wiley and Sons, Inc., NY).

Definitions

Unless otherwise indicated, the following terms are intended to have themeaning set forth below. Other terms are defined elsewhere throughoutthe specification.

Activate: As used herein the term “activate” is used in reference to areceptor or receptor complex to reflect a biological effect, directlyand/or by participation in a multicomponent signaling cascade, arisingfrom the binding of an agonist ligand to a receptor responsive to thebinding of the ligand.

Activity: As used herein, the term “activity” is used with respect to amolecule to describe a property of the molecule with respect to a testsystem (e.g., an assay) or biological or chemical property (e.g., thedegree of binding of the molecule to another molecule) or of a physicalproperty of a material or cell (e.g., modification of cell membranepotential). Examples of such biological functions include but are notlimited to catalytic activity of a biological agent, the ability tostimulate intracellular signaling, gene expression, cell proliferation,the ability to modulate immunological activity such as inflammatoryresponse. “Activity” is typically expressed as a level of a biologicalactivity per unit of agent tested such as [catalytic activity]/[mgprotein], [immunological activity]/[mg protein], international units(IU) of activity, [STAT5 phosphorylation]/[mg protein],[proliferation]/[mg protein], plaque forming units (pfu), etc. As usedherein, the term proliferative activity refers to an activity thatpromotes cell proliferation and replication, including dysregulated celldivision such as that observed in neoplastic diseases, inflammatorydiseases, fibrosis, dysplasia, cell transformation, metastasis, andangiogenesis.

Administer/Administration: The terms “administration” and “administer”are used interchangeably herein to refer the act of contacting asubject, including contacting a cell, tissue, organ, or biological fluidof the subject in vitro, in vivo or ex vivo with an agent (e.g., an aIFNgR2 binding molecule or an engineered cell expressing an IFNgR2binding molecule, a chemotherapeutic agent, an antibody, or apharmaceutical formulation comprising one or more of the foregoing).Administration of an agent may be achieved through any of a variety ofart recognized methods including but not limited to the topicaladministration, intravascular injection (including intravenous orintraarterial infusion), intradermal injection, subcutaneous injection,intramuscular injection, intraperitoneal injection, intracranialinjection, intratumoral injection, transdermal, transmucosal,iontophoretic delivery, intralymphatic injection, intragastric infusion,intraprostatic injection, intravesical infusion (e.g., bladder),inhalation (e.g respiratory inhalers including dry-powder inhalers),intraocular injection, intraabdominal injection, intralesionalinjection, intraovarian injection, intracerebral infusion or injection,intracerebroventricular injection (ICVI), and the like. The term“administration” includes contact of an agent to the cell, tissue ororgan as well as the contact of an agent to a fluid, where the fluid isin contact with the cell, tissue or organ.

Affinity: As used herein the term “affinity” refers to the degree ofspecific binding of a first molecule (e.g., a ligand) to a secondmolecule (e.g., a receptor) and is measured by the equilibriumdissociation constant K_(D), a ratio of the dissociation rate constantbetween the molecule and the its target (k_(off)) and the associationrate constant between the molecule and its target (k_(on)).

Agonist: As used herein, the term “agonist” refers to a first agent thatspecifically binds a second agent (“target”) and interacts with thetarget to cause or promote an increase in the activation of the target.In some instances, agonists are activators of receptor proteins thatmodulate cell activation, enhance activation, sensitize cells toactivation by a second agent, or up-regulate the expression of one ormore genes, proteins, ligands, receptors, biological pathways, that mayresult in cell proliferation or pathways that result in cell cyclearrest or cell death such as by apoptosis. In some embodiments, anagonist is an agent that binds to a receptor and alters the receptorstate resulting in a biological response that mimics the effect of theendogenous ligand of the receptor. The term “agonist” includes partialagonists, full agonists and superagonists. An agonist may be describedas a “full agonist” when such agonist which leads to a substantiallyfull biological response (i.e. the response associated with thenaturally occurring ligand/receptor binding interaction) induced byreceptor under study, or a partial agonist. A “superagonist” is a typeof agonist that can produce a maximal response greater than theendogenous agonist for the target receptor, and thus has an activity ofmore than 100% of the native ligand. A super agonist is typically asynthetic molecule that exhibits greater than 110%, alternativelygreater than 120%, alternatively greater than 130%, alternativelygreater than 140%, alternatively greater than 150%, alternativelygreater than 160%, or alternatively greater than 170% of the response inan evaluable quantitative or qualitative parameter of the naturallyoccurring form of the molecule when evaluated at similar concentrationsin a comparable assay. It should be noted that the biological effectsassociated with the full agonist may differ in degree and/or in kindfrom those biological effects of partial or superagonists. In contrastto agonists, antagonists may specifically bind to a receptor but do notresult the signal cascade typically initiated by the receptor and may tomodify the actions of an agonist at that receptor. Inverse agonists areagents that produce a pharmacological response that is opposite indirection to that of an agonist.

Antagonist: As used herein, the term “antagonist” or “inhibitor” refersto a molecule that opposes the action(s) of an agonist. An antagonistprevents, reduces, inhibits, or neutralizes the activity of an agonist,and an antagonist can also prevent, inhibit, or reduce constitutiveactivity of a target, e.g., a target receptor, even where there is noidentified agonist. Inhibitors are molecules that decrease, block,prevent, delay activation, inactivate, desensitize, or down-regulate,e.g., a gene, protein, ligand, receptor, biological pathway including animmune checkpoint pathway, or cell.

Antibody: As used herein, the term “antibody” refers collectively to:(a) a glycosylated or non-glycosylated immunoglobulin that specificallybinds to target molecule, and (b) immunoglobulin derivatives thereof,including but not limited to antibody fragments such as single domainantibodies. In some embodiments the immunoglobulin derivative competeswith the immunoglobulin from which it was derived for binding to thetarget molecule. The term antibody is not restricted to immunoglobulinsderived from any particular species and includes murine, human, equine,camelids, antibodies of cartilaginous fishes including, but not limitedto, sharks. The term “antibody” encompasses antibodies isolatable fromnatural sources or from animals following immunization with an antigenand as well as engineered antibodies including monoclonal antibodies,bispecific antibodies, tri-specific, chimeric antibodies, humanizedantibodies, human antibodies, CDR-grafted, veneered, or deimmunized(e.g., to remove T-cell epitopes) antibodies, camelized (in the case ofVHHs), or molecules comprising binding domains of antibodies (e.g.,CDRs) in non-immunoglobulin scaffolds. The term “antibody” should not beconstrued as limited to any particular means of synthesis and includesnaturally occurring antibodies isolatable from natural sources and aswell as engineered antibodies molecules that are prepared by“recombinant” means including antibodies isolated from transgenicanimals that are transgenic for human immunoglobulin genes or ahybridoma prepared therefrom, antibodies isolated from a host celltransformed with a nucleic acid construct that results in expression ofan antibody, antibodies isolated from a combinatorial antibody libraryincluding phage display libraries. In one embodiment, an “antibody” is amammalian immunoglobulin of the IgG1, IgG2, IgG3 or IgG4 class. In someembodiments, the antibody is a “full length antibody” comprisingvariable and constant domains providing binding and effector functions.The term “single domain antibody” (sdAb) as used herein refers anantibody fragment consisting of a monomeric variable antibody domainthat is able to bind specifically to an antigen and compete for bindingwith the parent antibody from which it is derived. The term “singledomain antibody” includes scFv and VHH molecules. As used herein, theterm “VHH” refers to a single domain antibody derived from camelidantibody typically obtained from immunization of camelids (includingcamels, llamas and alpacas (see, e.g., Hamers-Casterman, et al. (1993)Nature 363:446-448). VHHs are also referred to as heavy chain antibodiesor Nanobodies® as Single domain antibodies may also be derived fromnon-mammalian sources such as VHHs obtained from IgNAR antibodiesimmunization of cartilaginous fishes including, but not limited to,sharks.

Biological Sample: As used herein, the term “biological sample” or“sample” refers to a sample obtained (or derived) from a subject. By wayof example, a biological sample comprises a material selected from thegroup consisting of body fluids, blood, whole blood, plasma, serum,mucus secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolarlavage fluid (BALF), fluids of the eye (e.g., vitreous fluid, aqueoushumor), lymph fluid, lymph node tissue, spleen tissue, bone marrow,tumor tissue, including immunoglobulin enriched or cell-type specificenriched fractions derived from one or more of such tissues.

IFNgR2 cell: The terms “IFNgR2 cell”, “IFNgR2-expressing cell”,“IFNgR2-positive cell” and “IFNgR2+” cell are used interchangeablyherein to refer to a cell which expresses and displays the IFNgR2antigen on the extracellular surface of the cell membrane. Similarly,the terms “IFNgR2-negative cell”, “IFNgR2—cells” as are usedinterchangeably herein to describe cells which do not express or displayIFNgR2 antigen on the cell surface.

CDR: As used herein, the term “CDR” or “complementarity determiningregion” is intended to mean the non-contiguous antigen combining sitesfound within the variable region of both heavy and light chainimmunoglobulin polypeptides. CDRs have been described by Kabat et al., JBiol. Chem. 252:6609-6616 (1977); Kabat, et al., U.S. Dept. of Healthand Human Services publication entitled “Sequences of proteins ofimmunological interest” (1991) (also referred to herein as “Kabat 1991”or “Kabat”); by Chothia, et al. (1987) J. Mol. Biol. 196:901-917 (alsoreferred to herein as “Chothia”); and MacCallum, et al. (1996) J. Mol.Biol. 262:732-745, where the definitions include overlapping or subsetsof amino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orgrafted antibodies or variants thereof is intended to be within thescope of the term as defined and used herein. The term “ChothiaNumbering” as used herein is recognized in the arts and refers to asystem of numbering amino acid residues based on the location of thestructural loop regions (Chothiaet al. 1986, Science 233:755-758;Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB227:799-817). For purposes of the present disclosure, unless otherwisespecifically identified, the positioning of CDRs2 and 3 in the variableregion of an antibody follows Kabat numbering or simply, “Kabat.” Thepositioning of CDR1 in the variableregion of an antibody followsa hybridof Kabat and Chothia numbering schemes.

Clonotype: A clonotype is defined as a collection of binding moleculesthat originate from the same B-cell progenitor cell. The term“clonotype” is used herein to refer to a collection of antigen bindingmolecules that belong to the same germline family, have the same CDR3lengths, and have 70% or greater homology in CDR3 sequence

Comparable: As used herein, the term “comparable” is used to describethe degree of difference in two measurements of an evaluablequantitative or qualitative parameter. For example, where a firstmeasurement of an evaluable quantitative parameter and a secondmeasurement of the evaluable parameter do not deviate beyond a rangethat the skilled artisan would recognize as not producing astatistically significant difference in effect between the two resultsin the circumstances, the two measurements would be considered“comparable.” In some instances, measurements may be considered“comparable” if one measurement deviates from another by less than 35%,alternatively by less than 30%, alternatively by less than 25%,alternatively by less than 20%, alternatively by less than 15%,alternatively by less than 10%, alternatively by less than 7%,alternatively by less than 5%, alternatively by less than 4%,alternatively by less than 3%, alternatively by less than 2%, or by lessthan 1%. In particular embodiments, one measurement is comparable to areference standard if it deviates by less than 15%, alternatively byless than 10%, or alternatively by less than 5% from the referencestandard.

Conservative Amino Acid Substitution: As used herein, the term“conservative amino acid substitution” refers to an amino acidreplacement that changes a given amino acid to a different amino acidwith similar biochemical properties (e.g., charge, hydrophobicity, andsize). For example, the amino acids in each of the following groups canbe considered as conservative amino acids of each other: (1) hydrophobicamino acids: alanine, isoleucine, leucine, tryptophan, phenylalanine,valine, proline, and glycine; (2) polar amino acids: glutamine,asparagine, histidine, serine, threonine, tyrosine, methionine, andcysteine; (3) basic amino acids: lysine and arginine; and (4) acidicamino acids: aspartic acid and glutamic acid.

Derived From: As used herein in the term “derived from”, in the contextof an amino acid sequence is meant to indicate that the polypeptide ornucleic acid has a sequence that is based on that of a referencepolypeptide or nucleic acid and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made. By way ofexample, the term “derived from” includes homologs or variants ofreference amino acid or DNA sequences.

Effective Concentration (EC): As used herein, the terms “effectiveconcentration” or its abbreviation “EC” are used interchangeably torefer to the concentration of an agent in an amount sufficient to effecta change in a given parameter in a test system. The abbreviation “E”refers to the magnitude of a given biological effect observed in a testsystem when that test system is exposed to a test agent. When themagnitude of the response is expressed as a factor of the concentration(“C”) of the test agent, the abbreviation “EC” is used. In the contextof biological systems, the term Emax refers to the maximal magnitude ofa given biological effect observed in response to a saturatingconcentration of an activating test agent. When the abbreviation EC isprovided with a subscript (e.g., EC₄₀, EC₅₀, etc.) the subscript refersto the percentage of the Emax of the biological response observed atthat concentration. For example, the concentration of a test agentsufficient to result in the induction of a measurable biologicalparameter in a test system that is 30% of the maximal level of suchmeasurable biological parameter in response to such test agent, this isreferred to as the “EC₃₀” of the test agent with respect to suchbiological parameter. Similarly, the term “EC₁₀₀” is used to denote theeffective concentration of an agent that results the maximal (100%)response of a measurable parameter in response to such agent. Similarly,the term EC₅₀ (which is commonly used in the field of pharmacodynamics)refers to the concentration of an agent sufficient to results in thehalf-maximal (about 50%) change in the measurable parameter. The term“saturating concentration” refers to the maximum possible quantity of atest agent that can dissolve in a standard volume of a specific solvent(e.g., water) under standard conditions of temperature and pressure. Inpharmacodynamics, a saturating concentration of a drug is typically usedto denote the concentration sufficient of the drug such that allavailable receptors are occupied by the drug, and EC₅₀ is the drugconcentration to give the half-maximal effect.

Enriched: As used herein in the term “enriched” refers to a sample thatis non-naturally manipulated so that a species (e.g., a molecule orcell) of interest is present in: (a) a greater concentration (e.g., atleast 3-fold greater, alternatively at least 5-fold greater,alternatively at least 10-fold greater, alternatively at least 50-foldgreater, alternatively at least 100-fold greater, or alternatively atleast 1000-fold greater) than the concentration of the species in thestarting sample, such as a biological sample (e.g., a sample in whichthe molecule naturally occurs or in which it is present afteradministration); or (b) a concentration greater than the environment inwhich the molecule was made (e.g., a recombinantly modified bacterial ormammalian cell).

Extracellular Domain: As used herein the term “extracellular domain” orits abbreviation “ECD” refers to the portion of a cell surface protein(e.g., a cell surface receptor) which is external to of the plasmamembrane of a cell. The cell surface protein may be transmembraneprotein, a cell surface or membrane associated protein.

Identity: The term “identity,” as used herein in reference topolypeptide or DNA sequences, refers to the subunit sequence identitybetween two molecules. When a subunit position in both of the moleculesis occupied by the same monomeric subunit (i.e., the same amino acidresidue or nucleotide), then the molecules are identical at thatposition. The similarity between two amino acid or two nucleotidesequences is a direct function of the number of identical positions. Ingeneral, the sequences are aligned so that the highest order match isobtained. If necessary, identity can be calculated using publishedtechniques and widely available computer programs, such as BLAST 2.0algorithms, which are described in Altschul et al. (1990) J Mol. Biol.215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25:3389-3402. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (NCBI) website. The algorithm involves first identifying high scoring sequencepairs (HSPs) by identifying short words of length W of the querysequence, which either match or satisfy some positive-valued thresholdscore “T” when aligned with a word of the same length in a databasesequence. T is referred to as the neighborhood word score threshold(Altschul, et al., supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters“M” (the reward score for a pair of matching residues; always >0) and“N” (the penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: (a)the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or (b) the end of either sequence is reached. The BLAST algorithmparameters “W”, “T”, and “X” determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) functionssimilarly but uses as defaults a word size (“W”) of 28, an expectation(“E”) of 10, M=1, N=−2, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as defaults a word size (W) of 3, anexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &Henikoff, (1989) PNAS(USA) 89:10915-10919).

In An Amount Sufficient Amount to Cause a Response: As used herein thephrase “in an amount sufficient to cause a response” is used inreference to the amount of a test agent sufficient to provide adetectable change in the level of an indicator measured before (e.g., abaseline level) and after the application of a test agent to a testsystem. In some embodiments, the test system is a cell, tissue ororganism. In some embodiments, the test system is an in vitro testsystem such as a fluorescent assay. In some embodiments, the test systemis an in vivo system which involves the measurement of a change in thelevel a parameter of a cell, tissue, or organism reflective of abiological function before and after the application of the test agentto the cell, tissue, or organism. In some embodiments, the indicator isreflective of biological function or state of development of a cellevaluated in an assay in response to the administration of a quantity ofthe test agent. In some embodiments, the test system involves themeasurement of a change in the level an indicator of a cell, tissue, ororganism reflective of a biological condition before and after theapplication of one or more test agents to the cell, tissue, or organism.The term “in an amount sufficient to effect a response” may besufficient to be a therapeutically effective amount but may also be moreor less than a therapeutically effective amount.

In Need of Treatment: The term “in need of treatment” as used hereinrefers to a judgment made by a physician or other caregiver with respectto a subject that the subject requires or will potentially benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of the physician's or caregiver's expertise.

In Need of Prevention: As used herein the term “in need of prevention”refers to a judgment made by a physician or other caregiver with respectto a subject that the subject requires or will potentially benefit frompreventative care. This judgment is made based upon a variety of factorsthat are in the realm of a physician's or caregiver's expertise.

Inhibitor: As used herein the term “inhibitor” refers to a molecule thatdecreases, blocks, prevents, delays activation of, inactivates,desensitizes, or down-regulates, e.g., a gene, protein, ligand,receptor, or cell. An inhibitor can also be defined as a molecule thatreduces, blocks, or inactivates a constitutive activity of a cell ororganism.

Intracellular Domain: As used herein the term “intracellular domain” orits abbreviation “ICD” refers to the portion of a cell surface protein(e.g., a cell surface receptor) which is inside of the plasma membraneof a cell. The ICD may include the entire cytoplasmic portion of atransmembrane protein or membrane associated protein, or intracellularprotein.

Isolated: As used herein the term “isolated” is used in reference to apolypeptide of interest that, if naturally occurring, is in anenvironment different from that in which it can naturally occur.“Isolated” is meant to include polypeptides that are within samples thatare substantially enriched for the polypeptide of interest and/or inwhich the polypeptide of interest is partially or substantiallypurified. Where the polypeptide is not naturally occurring, “isolated”indicates that the polypeptide has been separated from an environment inwhich it was synthesized, for example isolated from a recombinant cellculture comprising cells engineered to express the polypeptide or by asolution resulting from solid phase synthetic means.

Kabat Numbering: The term “Kabat numbering” as used herein is recognizedin the art and refers to a system of numbering amino acid residues whichare more variable than other amino acid residues (e.g., hypervariable)in the heavy and light chain regions of immunoglobulins (Kabat, et al.,(1971) Ann. NY Acad. Sci. 190:382-93; Kabat, et al., (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242). For purposes ofthe present disclosure, unless otherwise specifically identified, thepositioning of CDRs in the variable region of an antibody follows Kabatnumbering or simply, “Kabat.”

Ligand: As used herein, the term “ligand” refers to a molecule thatspecifically binds a receptor and causes a change in the receptor so asto effect a change in the activity of the receptor or a response in cellthat expresses that receptor. In one embodiment, the term “ligand”refers to a molecule or complex thereof that can act as an agonist orantagonist of a receptor. As used herein, the term “ligand” encompassesnatural and synthetic ligands. “Ligand” also encompasses smallmolecules, peptide mimetics of cytokines and antibodies. The complex ofa ligand and receptor is termed a “ligand-receptor complex.” A ligandmay comprise one domain of a polyprotein or fusion protein (e.g., eitherdomain of an antibody/ligand fusion protein).

Modulate: As used herein, the terms “modulate”, “modulation” and thelike refer to the ability of a test agent to cause a response, eitherpositive or negative or directly or indirectly, in a system, including abiological system, or biochemical pathway. The term modulator includesboth agonists (including partial agonists, full agonists andsuperagonists) and antagonists.

Nucleic Acid: The terms “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

Operably Linked: The term “operably linked” is used herein to refer tothe relationship between molecules, typically polypeptides or nucleicacids, which are arranged in a construct such that each of the functionsof the component molecules is retained although the operable linkage mayresult in the modulation of the activity, either positively ornegatively, of the individual components of the construct. For example,the operable linkage of a polyethylene glycol (PEG) molecule to awild-type protein may result in a construct where the biologicalactivity of the protein is diminished relative to the to the wild-typemolecule, however the two are nevertheless considered operably linked.When the term “operably linked” is applied to the relationship ofmultiple nucleic acid sequences encoding differing functions, themultiple nucleic acid sequences when combined into a single nucleic acidmolecule that, for example, when introduced into a cell usingrecombinant technology, provides a nucleic acid which is capable ofeffecting the transcription and/or translation of a particular nucleicacid sequence in a cell. For example, the nucleic acid sequence encodinga signal sequence may be considered operably linked to DNA encoding apolypeptide if it results in the expression of a preprotein whereby thesignal sequence facilitates the secretion of the polypeptide; a promoteror enhancer is considered operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isconsidered operably linked to a coding sequence if it is positioned soas to facilitate translation. Generally, in the context of nucleic acidmolecules, the term “operably linked” means that the nucleic acidsequences being linked are contiguous, and, in the case of a secretoryleader or associated subdomains of a molecule, contiguous and in readingphase. However, certain genetic elements such as enhancers may functionat a distance and need not be contiguous with respect to the sequence towhich they provide their effect but nevertheless may be consideredoperably linked.

Parent Polypeptide: As used herein, the terms “parent polypeptide” or“parent protein” are used interchangeably to designate the source of asecond polypeptide (e.g., a derivative, mutein or variant) which ismodified with respect to a first “parent” polypeptide. In someinstances, the parent polypeptide is a wild-type or naturally occurringform of a protein. In some instance, the parent polypeptide may be amodified form a naturally occurring protein that is further modified.The term “parent polypeptide” may refer to the polypeptide itself orcompositions that comprise the parent polypeptide (e.g., glycosylated orPEGylated forms and/or fusion proteins comprising the parentpolypeptide).

Partial Agonist: As used herein, the term “partial agonist” refers to amolecule that specifically binds that bind to and activate a givenreceptor but possess only partial activation the receptor relative to afull agonist. Partial agonists may display both agonistic andantagonistic effects. For example, when both a full agonist and partialagonist are present, the partial agonist acts as a competitiveantagonist by competing with the full agonist for the receptor bindingresulting in net decrease in receptor activation relative to the contactof the receptor with the full agonist in the absence of the partialagonist. Partial agonists can be used to activate receptors to give adesired submaximal response in a subject when inadequate amounts of theendogenous ligand are present, or they can reduce the overstimulation ofreceptors when excess amounts of the endogenous ligand are present. Themaximum response (Emax) produced by a partial agonist is called itsintrinsic activity and may be expressed on a percentage scale where afull agonist produced a 100% response. An partial agonist may havegreater than 10% but less than 100%, alternatively greater than 20% butless than 100%, alternatively greater than 30% but less than 100%,alternatively greater than 40% but less than 100%, alternatively greaterthan 50% but less than 100%, alternatively greater than 60% but lessthan 100%, alternatively greater than 70% but less than 100%,alternatively greater than 80% but less than 100%, or alternativelygreater than 90% but less than 100%, of the activity of the referencepolypeptide when evaluated at similar concentrations in a given assaysystem.

Polypeptide: As used herein the terms “polypeptide,” “peptide,” and“protein”, used interchangeably herein, refer to a polymeric form ofamino acids of any length, which can include genetically coded andnon-genetically coded amino acids, chemically or biochemically modifiedor derivatized amino acids, and polypeptides having modified polypeptidebackbones. The term polypeptide include fusion proteins, including, butnot limited to, fusion proteins with a heterologous amino acid sequence;fusion proteins with heterologous and homologous leader sequences;fusion proteins with or without N-terminal methionine residues; fusionproteins with amino acid sequences that facilitate purification such aschelating peptides; fusion proteins with immunologically taggedproteins; fusion proteins comprising a peptide with immunologicallyactive polypeptide fragment (e.g., antigenic diphtheria or tetanus toxinor toxoid fragments) and the like.

Prevent: As used herein the terms “prevent”, “preventing”, “prevention”and the like refer to a course of action initiated with respect to asubject prior to the onset of a disease, disorder, condition or symptomthereof so as to prevent, suppress, inhibit or reduce, eithertemporarily or permanently, a subject's risk of developing a disease,disorder, condition or the like (as determined by, for example, theabsence of clinical symptoms) or delaying the onset thereof. A course ofaction to prevent a disease, disorder or condition in a subject istypically applied in the context of a subject who is predisposed todeveloping a disease, disorder or condition due to genetic, experientialor environmental factors of developing a particular disease, disorder orcondition. In certain instances, the terms “prevent”, “preventing”,“prevention” are also used to refer to the slowing of the progression ofa disease, disorder or condition from an existing state to a moredeleterious state.

Receptor: As used herein, the term “receptor” refers to a polypeptidehaving a domain that specifically binds a ligand that binding of theligand results in a change to at least one biological property of thepolypeptide. In some embodiments, the receptor is a cell membraneassociated protein that comprises and extracellular domain (ECD) and amembrane associated domain which serves to anchor the ECD to the cellsurface. In some embodiments of cell surface receptors, the receptor isa membrane spanning polypeptide comprising an intracellular domain (ICD)and extracellular domain (ECD) linked by a membrane spanning domaintypically referred to as a transmembrane domain (TM). The binding of acognate ligand to the receptor results in a conformational change in thereceptor resulting in a measurable biological effect. In some instances,where the receptor is a membrane spanning polypeptide comprising an ECD,TM and ICD, the binding of the ligand to the ECD results in a measurableintracellular biological effect mediated by one or more domains of theICD in response to the binding of the ligand to the ECD. In someembodiments, a receptor is a component of a multi-component complex tofacilitate intracellular signaling. For example, the ligand may bind acell surface receptor that is not associated with any intracellularsignaling alone but upon ligand binding facilitates the formation of aheteromultimeric (including heterodimeric, heterotrimeric, etc.) orhomomultimeric (including homodimeric, homotrimeric, homotetrameric,etc.) complex that results in a measurable biological effect in the cellsuch as activation of an intracellular signaling cascade (e.g., theJak/STAT pathway). In some embodiments, a receptor is a membranespanning single chain polypeptide comprising ECD, TM and ICD domainswherein the ECD, TM and ICD domains are derived from the same ordiffering naturally occurring receptor variants or synthetic functionalequivalents thereof.

Recombinant: As used herein, the term “recombinant” is used as anadjective to refer to the method by which a polypeptide, nucleic acid,or cell was modified using recombinant DNA technology. A “recombinantprotein” is a protein produced using recombinant DNA technology and isfrequently abbreviated with a lower case “r” preceding the protein nameto denote the method by which the protein was produced (e.g.,recombinantly produced human growth hormone is commonly abbreviated“rhGH”). Similarly a cell is referred to as a “recombinant cell” if thecell has been modified by the incorporation (e.g., transfection,transduction, infection) of exogenous nucleic acids (e.g., ssDNA, dsDNA,ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids andthe like) using recombinant DNA technology. The techniques and protocolsfor recombinant DNA technology are well known in the art such as thosecan be found in Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.)and other standard molecular biology laboratory manuals.

Response: The term “response,” for example, of a cell, tissue, organ, ororganism, encompasses a quantitative or qualitative change in aevaluable biochemical or physiological parameter, (e.g., concentration,density, adhesion, proliferation, activation, phosphorylation,migration, enzymatic activity, level of gene expression, rate of geneexpression, rate of energy consumption, level of or state ofdifferentiation) where the change is correlated with the activation,stimulation, or treatment, with or contact with exogenous agents orinternal mechanisms such as genetic programming. In certain contexts,the terms “activation”, “stimulation”, and the like refer to cellactivation as regulated by internal mechanisms, as well as by externalor environmental factors; whereas the terms “inhibition”,“down-regulation” and the like refer to the opposite effects. A“response” may be evaluated in vitro such as through the use of assaysystems, surface plasmon resonance, enzymatic activity, massspectroscopy, amino acid or protein sequencing technologies. A“response” may be evaluated in vivo quantitatively by evaluation ofobjective physiological parameters such as body temperature, bodyweight,tumor volume, blood pressure, results of X-ray or other imagingtechnology or qualitatively through changes in reported subjectivefeelings of well-being, depression, agitation, or pain. In someembodiments, the level of proliferation of CD3 activated primary humanT-cells may be evaluated in a bioluminescent assay that generates aluminescent signal that is proportional to the amount of ATP presentwhich is directly proportional to the number of cells present in cultureas described in Crouch, et al. (1993) J. Immunol. Methods 160: 81-8 orusing commercially available assays such as the CellTiter-Glo® 2.0 CellViability Assay or CellTiter-Glo® 3D Cell Viability kits commerciallyavailable from Promega Corporation, Madison Wis. 53711 as catalognumbers G9241 and G9681 in substantial accordance with the instructionsprovided by the manufacturer. In some embodiments, the level ofactivation of T cells in response to the administration of a test agentmay be determined by flow cytometric methods as described as determinedby the level of STAT (e.g., STAT1, STAT3, STAT5) phosphorylation inaccordance with methods well known in the art. For example, STAT5phosphorylation may be measured using flow cytometric techniques asdescribed in Horta, et al. supra., Garcia, et al., supra, orcommercially available kits such as the Phospho-STAT5 (Tyr694) kit(commercially available from Perkin-Elmer, Waltham Mass. as Part Number64AT5PEG) in performed in substantial accordance with the instructionsprovided by the manufacturer.

Significantly Reduced Binding: As used herein, the term “exhibitssignificantly reduced binding” is used with respect a variant of a firstmolecule (e.g., a ligand or antibody) which exhibits a significantreduction in the affinity for a second molecule (e.g., receptor orantigen) relative the parent form of the first molecule. With respect toantibody variants, an antibody variant “exhibits significantly reducedbinding” if the affinity of the variant antibody for an antigen if thevariant binds to the native form of the receptor with and affinity ofless than 20%, alternatively less than about 10%, alternatively lessthan about 8%, alternatively less than about 6%, alternatively less thanabout 4%, alternatively less than about 2%, alternatively less thanabout 1%, or alternatively less than about 0.5% of the parent antibodyfrom which the variant was derived. Similarly, with respect to variantligands, a variant ligand “exhibits significantly reduced binding” ifthe affinity of the variant ligand binds to a receptor with an affinityof less than 20%, alternatively less than about 10%, alternatively lessthan about 8%, alternatively less than about 6%, alternatively less thanabout 4%, alternatively less than about 2%, alternatively less thanabout 1%, or alternatively less than about 0.5% of the parent ligandfrom which the variant ligand was derived. Similarly, with respect tovariant receptors, a variant ligand “exhibits significantly reducedbinding” if the affinity of the variant receptors binds to a with anaffinity of less than 20%, alternatively less than about 10%,alternatively less than about 8%, alternatively less than about 6%,alternatively less than about 4%, alternatively less than about 2%,alternatively less than about 1%, or alternatively less than about 0.5%of the parent receptor from which the variant receptor was derived.

Small Molecule(s): The term “small molecules” refers to chemicalcompounds (typically pharmaceutically active compounds) having amolecular weight that is less than about 10 kDa, less than about 2 kDa,or less than about 1 kDa. Small molecules include, but are not limitedto, inorganic molecules, organic molecules, organic molecules containingan inorganic component, molecules comprising a radioactive atom, andsynthetic molecules. The term “small molecule” is a term well understoodto those of ordinary skill in the pharmaceutical arts and is typicallyused to distinguish organic chemical compounds from biologics.

Specifically Binds: As used herein the term “specifically binds” refersto the degree of affinity for which a first molecule exhibits withrespect to a second molecule. In the context of binding pairs (e.g.,ligand/receptor, antibody/antigen) a first molecule of a binding pair issaid to specifically bind to a second molecule of a binding pair whenthe first molecule of the binding pair does not bind in a significantamount to other components present in the sample. A first molecule of abinding pair is said to specifically bind to a second molecule of abinding pair when the first molecule of the binding pair when theaffinity of the first molecule for the second molecule is at leasttwo-fold greater, alternatively at least five times greater,alternatively at least ten times greater, alternatively at least20-times greater, or alternatively at least 100-times greater than theaffinity of the first molecule for other components present in thesample. In a particular embodiment, where the first molecule of thebinding pair is an antibody, the antibody specifically binds to theantigen (or antigenic determinant (epitope) of a protein, antigen,ligand, or receptor) if the equilibrium dissociation constant (K_(D))between antibody and the antigen is lesser than about 10⁻⁶ M,alternatively lesser than about 10⁻⁸ M, alternatively lesser than about10⁻¹⁰ M, alternatively lesser than about 10⁻¹¹ M, lesser than about10⁻¹² M as determined by, e.g., Scatchard analysis (Munsen, et al.(1980) Analyt. Biochem. 107:220-239). In one embodiment where the ligandis an ILR binding sdAb and the receptor comprises an ILR, the ILRbinding sdAb specifically binds if the equilibrium dissociation constant(K_(D)) of the ILR binding sdAb/ILR ECD is lesser than about 10⁻⁵M,alternatively lesser than about 10⁻⁶ M, alternatively lesser than about10⁻⁷M, alternatively lesser than about 10⁻⁸M, alternatively lesser thanabout 10⁻⁹ M, alternatively lesser than about 10⁻¹⁰ M, or alternativelylesser than about 10⁻¹¹ M. Specific binding may be assessed usingtechniques known in the art including but not limited to competitionELISA assays, radioactive ligand binding assays (e.g., saturationbinding, Scatchard plot, nonlinear curve fitting programs andcompetition binding assays); non-radioactive ligand binding assays(e.g., fluorescence polarization (FP), fluorescence resonance energytransfer (FRET); liquid phase ligand binding assays (e.g., real-timepolymerase chain reaction (RT-qPCR), and immunoprecipitation); and solidphase ligand binding assays (e.g., multiwell plate assays, on-beadligand binding assays, on-column ligand binding assays, and filterassays)) and surface plasmon resonance assays (see, e.g., Drescher etal., (2009) Methods Mol Biol 493:323-343 with commercially availableinstrumentation such as the Biacore 8+, Biacore S200, Biacore T200 (GEHealthcare Bio-Sciences, 100 Results Way, Marlborough MA 01752). In someembodiments, the present disclosure provides molecules (e.g., ILRbinding sdAbs) that specifically bind to the hILR. As used herein, thebinding affinity of an ILR binding molecule for the ILR, the bindingaffinity may be determined and/or quantified by surface plasmonresonance (“SPR”). In evaluating binding affinity of an ILR bindingmolecule for the ILR, either member of the binding pair may beimmobilized, and the other element of the binding pair be provided inthe mobile phase. In some embodiments, the sensor chip on which theprotein of interest is to be immobilized is conjugated with a substanceto facilitate binding of the protein of interest such asnitrilotriacetic acid (NTA) derivatized surface plasmon resonance sensorchips (e.g., Sensor Chip NTA available from Cytiva Global Life ScienceSolutions USA LLC, Marlborough MA as catalog number BR100407), asanti-His tag antibodies (e.g. anti-histidine CM5 chips commerciallyavailable from Cytiva, Marlborough MA), protein A or biotin.Consequently, to evaluate binding, it is frequently necessary to modifythe protein to provide for binding to the substance conjugated to thesurface of the chip. For example, the one member of the binding pair tobe evaluated by incorporation of a chelating peptide comprisingpoly-histidine sequence (e.g., 6×His (SEQ ID NO: 165) or 8×His (SEQ IDNO: 166)) for retention on a chip conjugated with NTA. In someembodiments, the ILR binding molecule may be immobilized on the chip andILR (or ECD fragment thereof) be provided in the mobile phase.Alternatively, the ILR (or ECD fragment thereof) may be immobilized onthe chip and the ILR binding molecule be provided in the mobile phase.In either circumstance, it should be noted that modifications of someproteins for immobilization on a coated SPR chip may interfere with thebinding properties of one or both components of the binding pair to beevaluated by SPR. In such cases, it may be necessary to switch themobile and bound elements of the binding pair or use a chip with abinding agent that facilitates non-interfering conjugation of theprotein to be evaluated. Alternatively, when evaluating the bindingaffinity of ILR binding molecule for ILR using SPR, the ILR bindingmolecule may be derivatized by the C-terminal addition of a poly-Hissequence (e.g., 6×His (SEQ ID NO: 165) or 8×His (SEQ ID NO: 166)) andimmobilized on the NTA derivatized sensor chip and the ILR receptorsubunit for which the ILR VHH's binding affinity is being evaluated isprovided in the mobile phase. The means for incorporation of a poly-Hissequence into the C-terminus of the ILR binding molecule produced byrecombinant DNA technology is well known to those of skill in therelevant art of biotechnology. In some embodiments, the binding affinityof ILR binding molecule for an ILR comprises using SPR substantially inaccordance with the teaching of the Examples.

Subject: The terms “recipient”, “individual”, “subject”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.In some embodiments, the mammal is a human being.

Substantially Pure: As used herein, the term “substantially pure”indicates that a component of a composition makes up greater than about50%, alternatively greater than about 60%, alternatively greater thanabout 70%, alternatively greater than about 80%, alternatively greaterthan about 90%, alternatively greater than about 95% of the totalcontent of the composition. A protein that is “substantially pure”comprises greater than about 50%, alternatively greater than about 60%,alternatively greater than about 70%, alternatively greater than about80%, alternatively greater than about 90%, alternatively greater thanabout 95% of the total content of the composition.

Suffering From: As used herein, the term “suffering from” refers to adetermination made by a physician with respect to a subject based on theavailable objective or subjective information accepted in the field forthe identification of a disease, disorder or condition including but notlimited to X-ray, CT-scans, conventional laboratory diagnostic tests(e.g., blood count, etc.), genomic data, protein expression data,immunohistochemistry, that the subject requires or will benefit fromtreatment. The term suffering from is typically used in conjunction witha particular disease state such as “suffering from a neoplastic disease”refers to a subject which has been diagnosed with the presence of aneoplasm.

T-cell: As used herein the term “T-cell” or “T cell” is used in itsconventional sense to refer to a lymphocytes that differentiates in thethymus, possess specific cell-surface antigen receptors, and includesome that control the initiation or suppression of cell-mediated andhumoral immunity and others that lyse antigen-bearing cells. In someembodiments the T cell includes without limitation naïve CD8+ T cells,cytotoxic CD8+ T cells, naïve CD4+ T cells, helper T cells, e.g.,T_(H)1, T_(H)2, T_(H)9, T_(H)11, T_(H)22, T_(FH); regulatory T cells,e.g., TRI, Tregs, inducible Tregs; memory T cells, e.g., central memoryT cells, effector memory T cells, NKT cells, tumor infiltratinglymphocytes (TILs) and engineered variants of such T-cells including butnot limited to CAR-T cells, recombinantly modified TILs andTCR-engineered cells. In some embodiments the T cell is a T cellexpressing the IFNgR2 isoform referred to interchangeably as IFNgR2cell, IFNgR2+ cell, IFNgR2 T cell, or IFNgR2+ T cell).

Terminus/Terminal: As used herein in the context of the structure of apolypeptide, “N-terminus” (or “amino terminus”) and “C-terminus” (or“carboxyl terminus”) refer to the extreme amino and carboxyl ends of thepolypeptide, respectively, while the terms “N-terminal” and “C-terminal”refer to relative positions in the amino acid sequence of thepolypeptide toward the N-terminus and the C-terminus, respectively, andcan include the residues at the N-terminus and C-terminus, respectively.“Immediately N-terminal” refers to the position of a first amino acidresidue relative to a second amino acid residue in a contiguouspolypeptide sequence, the first amino acid being closer to theN-terminus of the polypeptide. “Immediately C-terminal” refers to theposition of a first amino acid residue relative to a second amino acidresidue in a contiguous polypeptide sequence, the first amino acid beingcloser to the C-terminus of the polypeptide.

Therapeutically Effective Amount: As used herein to the phrase“therapeutically effective amount” refers to the quantity of an agentwhen administered to a subject, either alone or as part of apharmaceutical composition or treatment regimen, in a single dose or aspart of a series of doses, provides a positive effect on anyquantitative or qualitative symptom, aspect, or characteristic of adisease, disorder or condition. A therapeutically effective amount canbe ascertained by measuring relevant physiological effects, and it maybe adjusted in connection with a dosing regimen and in response todiagnostic analysis of the subject's condition. The parameters forevaluation to determine a therapeutically effective amount of an agentare determined by the physician using art accepted diagnostic criteriaincluding but not limited to indicia such as age, weight, sex, generalhealth, ECOG score, observable physiological parameters, blood levels,blood pressure, electrocardiogram, computerized tomography, X-ray, andthe like. Alternatively, or in addition, other parameters commonlyassessed in the clinical setting may be monitored to determine if atherapeutically effective amount of an agent has been administered tothe subject such as body temperature, heart rate, normalization of bloodchemistry, normalization of blood pressure, normalization of cholesterollevels, or any symptom, aspect, or characteristic of the disease,disorder or condition, biomarkers (such as inflammatory cytokines,IFN-γ, granzyme, and the like), reduction in serum tumor markers,improvement in Response Evaluation Criteria In Solid Tumors (RECIST),improvement in Immune-Related Response Criteria (irRC), increase induration of survival, extended duration of progression free survival,extension of the time to progression, increased time to treatmentfailure, extended duration of event free survival, extension of time tonext treatment, improvement objective response rate, improvement in theduration of response, reduction of tumor burden, complete response,partial response, stable disease, and the like that that are relied uponby clinicians in the field for the assessment of an improvement in thecondition of the subject in response to administration of an agent. Inone embodiment, a therapeutically effective amount is an amount of anagent when used alone or in combination with another agent provides anprovides a positive effect on any quantitative or qualitative symptom,aspect, or characteristic of a disease, disorder or condition and doesnot result in non-reversible serious adverse events in the course ofadministration of the agent to the mammalian subject.

Transmembrane Domain: The term “transmembrane domain” or “TM” refers toa polypeptide domain of a membrane spanning polypeptide (e.g., atransmembrane receptor) which, when the membrane spanning polypeptide isassociated with a cell membrane, is which is embedded in the cellmembrane and is in peptidyl linkage with the extracellular domain (ECD)and the intracellular domain (ICD) of a membrane spanning polypeptide. Atransmembrane domain may be homologous (naturally associated with) orheterologous (not naturally associated with) with either or both of theextracellular and/or intracellular domains. In some embodiments, wherethe receptor is chimeric receptor comprising the intracellular domainderived from a first parental receptor and a second extracellulardomains are derived from a second different parental receptor, thetransmembrane domain of the chimeric receptor is the transmembranedomain normally associated with either the ICD or the ECD of the parentreceptor from which the chimeric receptor is derived.

Treat: The terms “treat”, “treating”, treatment” and the like refer to acourse of action (such as contacting the subject with pharmaceuticalcomposition comprising a IFNgR2 binding sdAb alone or in combinationwith a supplementary agent) that is initiated with respect to a subjectin response to a diagnosis that the subject is suffering from a disease,disorder or condition, or a symptom thereof, the course of action beinginitiated so as to eliminate, reduce, suppress, mitigate, or ameliorate,either temporarily or permanently, at least one of: (a) the underlyingcauses of such disease, disorder, or condition afflicting a subject;and/or (b) at least one of the symptoms associated with such disease,disorder, or condition. In some embodiments, treating includes a courseof action taken with respect to a subject suffering from a disease wherethe course of action results in the inhibition (e.g., arrests thedevelopment of the disease, disorder or condition or ameliorates one ormore symptoms associated therewith) of the disease in the subject.

Treg Cell or Regulatory T Cell. The terms “regulatory T cell”, “Tregcell”, or “Treg” are interchangeably herein to refers to a type of CD4⁺T cell that can suppress the responses of other T cells including butnot limited to effector T cells (T_(eff)). Treg cells are typicallycharacterized by expression of CD4 (CD4+), the CD25 subunit of the IL2receptor (CD25+), and the transcription factor forkhead box P3 (FOXP3+)(Sakaguchi, Annu Rev Immunol 22, 531-62 (2004). In some instances, theterm “conventional CD4⁺ T cells” is used to distinguish non-Treg CD4⁺ Tcells from CD4⁺ Tregs.

Variant: The terms “variant”, “protein variant” or “variant protein” or“variant polypeptide” are used interchangeably herein to refer to apolypeptide that differs from a parent polypeptide by virtue of at leastone amino acid modification, substitution, or deletion. The parentpolypeptide may be a naturally occurring or wild-type (WT) polypeptideor may be a modified version of a WT polypeptide. The term variantpolypeptide may refer to the polypeptide itself, a compositioncomprising the polypeptide, or the nucleic acid sequence that encodesit. In some embodiments, the variant polypeptide comprises from aboutone to about ten, alternatively about one to about eight, alternativelyabout one to about seven, alternatively about one to about five,alternatively about one to about four, alternatively from about one toabout three alternatively from one to two amino acid modifications,substitutions, or deletions, or alternatively a single amino acid aminoacid modification, substitution, or deletion compared to the parentpolypeptide. A variant may be at least about 99% identical,alternatively at least about 98% identical, alternatively at least about97% identical, alternatively at least about 95% identical, oralternatively at least about 90% identical to the parent polypeptidefrom which the variant is derived.

Wild Type: By “wild type” or “WT” or “native” herein is meant an aminoacid sequence or a nucleotide sequence that is found in nature,including allelic variations. A wild-type protein, polypeptide,antibody, immunoglobulin, IgG, etc. has an amino acid sequence or anucleotide sequence that has not been modified by the hand of man.

IFNgR2

The IFNgR2 binding molecules of the present disclosure specifically bindto the extracellular domain of the IFNgR2.

Human IFNgR2

In one embodiment, specifically bind to the extracellular domain of thehuman IFNgR2 receptor subunit (hIFNgR2). hIFNgR2 is expressed as a 337amino acid precursor comprising a 21 amino acid N-terminal signalsequence which is post-translationally cleaved to provide a 316 aminoacid mature protein. The canonical full-length acid hIFNgR2 precursor(including the signal peptide) is a 337 amino acid polypeptide havingthe amino acid sequence:

(SEQ ID NO: 1) MRPTLLWSLLLLLGVFAAAAAAPPDPLSQLPAPQHPKIRLYNAEQVLSWEPVALSNSTRPVVYQVQFKYTDSKWFTADIMSIGVNCTQITATECDFTAASPSAGFPMDFNVTLRLRAELGALHSAWVTMPWFQHYRNVTVGPPENIEVTPGEGSLIIRFSSPFDIADTSTAFFCYYVHYWEKGGIQQVKGPFRSNSISLDNLKPSRVYCLQVQAQLLWNKSNIFRVGHLSNISCYETMADASTELQQVILISVGTFSLLSVLAGACFFLVLKYRGLIKYWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPEKEQEDVLQTL

For purposes of the present disclosure, the numbering of amino acidresidues of the human IFNgR2 polypeptides as described herein is made inaccordance with the numbering of this canonical sequence (UniProtReference No. P38484, SEQ ID NO:1). Amino acids 1-21 of SEQ ID NO:1 areidentified as the signal peptide of hIFNgR2, amino acids 22-247 of SEQID NO:1 are identified as the extracellular domain, amino acids 248-268of SEQ ID NO:1 are identified as the transmembrane domain, and aminoacids 269-337 of SEQ ID NO:1 are identified as the intracellular domain.

For the purposes of generating antibodies that bind to the ECD ofIFNgR2, immunization may be performed with the extracellular domain ofthe hIFNgR2. The extracellular domain of hIFNgR2 is a 220 amino acidpolypeptide of the sequence:

(SEQ ID NO: 162) SQLPAPQHPKIRLYNAEQVLSWEPVALSNSTRPVVYQVQFKYTDSKWFTADIMSIGVNCTQITATECDFTAASPSAGFPMDFNVTLRLRAELGALHSAWVTMPWFQHYRNVTVGPPENIEVTPGEGSLIIRFSSPFDIADTSTAFFCYYVHYWEKGGIQQVKGPFRSNSISLDNLKPSRVYCLQVQAQLLWNKSNIFRVG HLSNISCYETMADASTELQQ.

Mouse IFNgR2

In one embodiment, specifically bind to the extracellular domain of themouse or murine IFNgR2 receptor subunit (mIFNgR2). mIFNgR2 is expressedas a 332 amino acid precursor comprising a 19 amino acid N-terminalsignal sequence which is post-translationally cleaved to provide a 313amino acid mature protein. The canonical full-length acid mIFNgR2precursor (including the 19 amino acid signal peptide) is a 332 aminoacid polypeptide having the amino acid sequence:

(SEQ ID NO: 163) MRPLPLWLPSLLLCGLGAAASSPDSFSQLAAPLNPRLHLYNDEQILTWEPSPSSNDPRPVVYQVEYSFIDGSWHRLLEPNCTDITETKCDLTGGGRLKLFPHPFTVFLRVRAKRGNLTSKWVGLEPFQHYENVTVGPPKNISVTPGKGSLVIHFSPPFDVFHGATFQYLVHYWEKSETQQEQVEGPFKSNSIVLGNLKPYRVYCLQTEAQLILKNKKIRPHGLLSNVSCHETTANASARLQQVILIPLGIFALLLGLTGACFTLFLKYQSRVKYWFQAPPNIPEQIEEYLKDPDQFILEVLDKDGSPKEDSWDSVSIISSPEKERDDVLQTP

For purposes of the present disclosure, the numbering of amino acidresidues of the mIFNgR2 polypeptides as described herein is made inaccordance with the numbering of this canonical sequence (UniProtReference No. Q63953, SEQ ID NO:163). Amino acids 1-19 of SEQ ID NO:163are identified as the signal peptide of mIFNgR2, amino acids 20-243 ofSEQ ID NO: 163 are identified as the extracellular domain, amino acids244-266 of SEQ ID NO:163 are identified as the transmembrane domain, andamino acids 267-332 of SEQ ID NO:163 are identified as the intracellulardomain.

For the purposes of generating antibodies that bind to the ECD ofIFNgR2, immunization may be performed with the extracellular domain ofthe mIFNgR2. The extracellular domain of the mIFNgR2 receptor is a 229amino acid polypeptide of the sequence:

(SEQ ID NO: 164) ASSPDSFSQLAAPLNPRLHLYNDEQILTWEPSPSSNDPRPVVYQVEYSFIDGSWHRLLEPNCTDITETKCDLTGGGRLKLFPHPFTVFLRVRAKRGNLTSKWVGLEPFQHYENVTVGPPKNISVTPGKGSLVIHFSPPFDVFHGATFQYLVHYWEKSETQQEQVEGPFKSNSIVLGNLKPYRVYCLQTEAQLILKNKKIRPHGLLSNVSCHETTANASARLQQV

Experimental

The single domain antibodies of the present disclosure were obtainedfrom camels by immunization with an extracellular domain of a IFNgR2receptor. IFNgR2 VHH molecules of the present disclosure of the presentdisclosure were generated in substantial accordance with the teaching ofthe Examples. Briefly, a camel was sequentially immunized with the ECDof the human IFNgR2 over a period several weeks of by the subcutaneousan adjuvanted composition containing a recombinantly produced fusionproteins comprising the extracellular domain of the IFNgR2, the humanIgG1 hinge domain and the human IgG1 heavy chain Fc. Followingimmunization, RNAs extracted from a blood sample of appropriate sizeVHH-hinge-CH2-CH3 species were transcribed to generate DNA sequences,digested to identify the approximately 400 bp fragment comprising thenucleic acid sequence encoding the VHH domain was isolated. The isolatedsequence was digested with restriction endonucleases to facilitateinsertion into a phagemid vector for in frame with a sequence encoding ahis-tag and transformed into E. coli to generate a phage library.Multiple rounds of biopanning of the phage library were conducted toidentify VHHs that bound to the ECD of IFNgR2. Individual phage cloneswere isolated for periplasmic extract ELISA (PE-ELISA) in a 96-wellplate format and selective binding confirmed by colorimetricdetermination. The IFNgR2 binding molecules that demonstrated specificbinding to the IFNgR2 antigen were isolated and sequenced and sequencesanalyzed to identify VHH sequences, CDRs and identify unique VHHclonotypes. As used herein, the term “clonotypes” refers a collection ofbinding molecules that originate from the same B-cell progenitor cell,in particular collection of antigen binding molecules that belong to thesame germline family, have the same CDR3 lengths, and have 70% orgreater homology in CDR3 sequence. The VHH molecules demonstratingspecific binding to the hIFNgR2 ECD antigen (anti-human IFNgR2 VHHs) andthe CDRs isolated from such VHHs are provided in Table 1. Nucleic acidsequences encoding the VHHs of Table 1 are provide in Table 2.

In some instances, due to sequence or structural similarities betweenthe extracellular domains of IFNgR2 receptors from various mammalianspecies, immunization with an antigen derived from a IFNgR2 of a firstmammalian species (e.g., the hIFNgR2-ECD) may provide antibodies whichspecifically bind to IFNgR2 receptors of one or more additionalmammalian species. Such antibodies are termed “cross reactive.” Forexample, immunization of a camelid with a human derived antigen (e.g.,the hIFNgR2-ECD) may generate antibodies that are cross-reactive themurine and human receptors. Evaluation of cross-reactivity of antibodywith respect to the receptors derived from other mammalian species maybe readily determined by the skilled artisan, for example using themethods relating to evaluation of binding affinity and/or specificbinding described elsewhere herein such as flow cytometry or SPR.

Consequently, the use of the term “human IFNgR2 VHH” or “hIFNgR2 VHH”merely denotes that the species of the IFNgR2 antigen used forimmunization of the camelid from which the VHH was derived was the humanIFNgR2 (e.g., the IFNgR2, ECD, SEQ ID NO:164 but should not beunderstood as limiting with respect to the specific binding affinity ofthe VHH for IFNgR2 molecules of other mammalian species. Similarly, theuse of the term “mouse IFNgR2 VHH” or “mIFNgR2 VHH” merely denotes thatthe species of the IFNgR2 antigen used for immunization of the camelidfrom which the VHH was derived was the murine IFNgR2 (e.g., the mIFNgR2ECD, SEQ ID NO:194) but should not be understood as limiting withrespect to the specific binding affinity of the VHH for IFNgR2 moleculesof other mammalian species.

The hIFNgR2 VHHs of Table 1 (SEQ ID NOS: 2-33) were evaluated forcross-reactivity with the mIFNgR2 by flow cytometry and were found tobind both the extracellular domain of hIFNgR2 (SEQ ID NO. 162) and theextracellular domain of mIFNgR2 (SEQ ID NO. 164). Consequently, the VHHsprovided in Table 1 may be used in both murine and human applicationsavoiding the necessity of a surrogate anti-mIFNgR2 for anti-hIFNgR2 forin vivo models of efficacy, such as a mouse model of a human diseasestate.

IFNgR2 Binding Molecules and Single Domain Antibodies

In some embodiments, a IFNgR2 binding molecule of the present disclosureis a single domain antibody (sdAb). The present disclosure relates toIFNgR2 binding molecules comprising single domain antibodies (sdAbs)that specifically bind to the extracellular domain of the human IFNgR2isoform (hIFNgR2) which are found on all IFNgR2-expressing cells.

A single-domain antibody (sdAb) is an antibody containing a singlemonomeric variable antibody domain. Like a full-length antibody, sdAbsare able to bind specifically to an antigenic determinant. hIFNgR2binding VHH single-domain antibodies can be engineered from heavy chainantibodies isolated from Camelidae mammals (e.g., camels, llamas,dromedary, alpaca, and guanaco) immunized with the extracellular domainof hIFNgR2 or an immunologically active fragment thereof. Descriptionsof sdAbs and VHHs can be found in, e.g., De Greve et al., (2019) CurrOpin Biotechnol. 61:96-101; Ciccarese, et al., (2019) Front Genet.10:997: Chanier and Chames (2019) Antibodies (Basel) 8(1); and DeVlieger, et al. (2018) Antibodies (Basel) 8(1). Alternatively, hIFNgR2single domain antibodies may be engineered from heavy chain antibodiesisolated from the IgNAR heavy chain antibodies isolated fromcartilaginous fishes immunized with the extracellular domain of hIFNgR2or an immunologically active fragment thereof hIFNgR2 binding sdAbs mayalso be obtained by splitting the dimeric variable domains fromimmunoglobulin G (IgG) isotypes from other mammalian species includinghumans, rats, rabbits immunized with the extracellular domain of hIFNgR2or an immunologically active fragment thereof. Although most researchinto sdAbs is currently based on heavy chain variable domains, sdAbsderived from light chains have also been shown to bind specifically tothe target proteins comprising the antigenic immunization sequence.Moller et al., J Biol Chem. 285(49):38348-38361, 2010.

In some embodiments, the sdAb is a VHH. A VHH is a type of sdAb that hasa single monomeric heavy chain variable antibody domain. Similar to atraditional antibody, a VHH is able to bind specifically to a specificantigen. An exemplary VHH has a molecular weight of approximately 12-15kDa which is much smaller than traditional mammalian antibodies (150-160kDa) composed of two heavy chains and two light chains. VHHs can befound in or produced from Camelidae mammals (e.g., camels, llamas,dromedary, alpaca, and guanaco) which are naturally devoid of lightchains.

The present disclosure provides IFNgR2 binding molecules comprising apolypeptide having at least 75%, alternatively 80%, alternatively 90%,alternatively 95%, alternatively 98%, or alternatively 99% or 100%identity to a polypeptide of any one of SEQ ID NOS:2-33.

Modified Forms of Single Domain Antibodies

CDR Grafted sdAbs

In some embodiments, the IFNgR2 binding sdAb of the present disclosureis a CDR grafted IFNgR2 binding sdAb. CDRs obtained from antibodies,heavy chain antibodies, and sdAbs derived therefrom may be grafted ontoalternative frameworks as described in Saerens, et al. (2005) J. MolBiol 352:597-607 to generate CDR-grafted sdAbs. In some embodiments, thepresent disclosure provides a IFNgR2 binding molecule comprising a CDRgrafted IFNgR2 binding sdAb, said CDR-grafted IFNgR2 binding sdAbcomprising a set of CDRs1, 2, and 3 as shown in a row of the Table 1above. In some embodiments, the present disclosure provides a IFNgR2binding molecule comprising a CDR grafted IFNgR2 binding sdAb, saidCDR-grafted IFNgR2 binding sdAb comprising a set of CDRs1, 2, and 3 asshown in a row of the Table 1 above.

Chimeric and Humanized sdAbs

Any framework region can be used with the CDRs as described herein. Insome embodiments, the IFNgR2 binding sdAb is a chimeric sdAb, in whichthe CDRs are derived from one species (e.g., camel) and the frameworkand/or constant regions are derived from another species (e.g., human ormouse). In specific embodiments, the framework regions are human orhumanized sequences. Thus, humanized IFNgR2 binding sdAbs derived fromhIFNgR2 binding VHHs are considered within the scope of the presentdisclosure. The techniques for humanization of camelid single domainantibodies are well known in the art. See, e.g., Vincke, et al. (2009)General Strategy to Humanize a Camelid Single-domain Antibody andIdentification of a Universal Humanized Nanobody Scaffold J. Biol. Chem.284(5)3273-3284.

In some embodiments, a VHH described herein can be humanized to containhuman framework regions. Examples of human germlines that could be usedto create humanized VHHs include, but are not limited to, VH3-23 (e.g.,UniProt ID: P01764), VH3-74 (e.g., UniProtID: A0A0B4J1X5), VH3-66 (e.g.,UniProtID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g.,UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782).

Elimination of N-Linked Glycosylation Sites

In some embodiments, it is possible that an amino acid sequence(particularly a CDR sequence) of the hIFNgR2 binding sdAb may contain aglycosylation motif, particularly an N-linked glycosylation motif of thesequence Asn-X-Ser (N—X-S) or Asn-X-Thr (N-X-T), wherein X is any aminoacid except for proline. In such instances, it is desirable to eliminatesuch N-linked glycosylation motifs by modifying the sequence of theN-linked glycosylation motif to prevent glycosylation. In someembodiments, the elimination of the Asn-X-Ser (N—X-S) N-linkedglycosylation motif may be achieved by the incorporation of conservativeamino acid substitution of the Asn (N) residue and/or Ser (S) residue ofthe Asn-X-Ser (N—X-S) N-linked glycosylation motif In some embodiments,the elimination of the Asn-X-Thr (N-X-T) N-linked glycosylation motifmay be achieved by the incorporation of conservative amino acidsubstitution of the Asn (N) residue and/or Thr (T) residue of theAsn-X-Thr (N-X-T) N-linked glycosylation motif In some embodiments,elimination of the glycosylation site is not required when the IFNgR2binding molecule is expressed in procaryotic host cells. Sinceprocaryotic cells do not provide a mechanism for glycosylation ofrecombinant proteins, when employing a procaryotic expression system toproduce a recombinant IFNgR2 binding sdAb the modification of thesequence to eliminate the N-linked glycosylation sites may be obviated.

IFNgR2 Binding Molecules Comprising Additional Agents

In some embodiments, a IFNgR2 binding molecule of the present disclosurea IFNgR2 single domain antibody (IFNgR2 sdAb) conjugated to one or moreadditional biologically active agents including but not limited to,therapeutic agents, chemically, optically or radioactively activeagents, including combinations thereof. The conjugation of at least onesuch biologically, chemically, optically or radioactively active agentconfer additional biological or chemical properties to IFNgR2 bindingsdAb, the combination providing a IFNgR2 binding molecule possessingadditional or alternative utilities.

For example, the additional agent may be a molecule selected from one ormore of: immunomodulatory agents (e.g., immunogens); molecules thatimprove aqueous solubility (e.g., water soluble polymers and hydrophilicmolecules such as sugars); carrier molecules that extend in vivohalf-life (e.g., PEGylation, Fc fusions or acylation); generation ofantibodies for use in detection assays (e.g., epitope tags), enhanceease of purification (e.g., chelating peptides such as poly-His tags);targeting domains that provide selective targeting IFNgR2 bindingmolecule to a particular cell or tissue type; therapeutic agents (e.g.,therapeutic agents including small molecule or polypeptide agents);agents that visibility to optical or electromagnetic sensors (e.g.,radionucleotides or fluorescent agents). In some embodiments, the linkeris a cleavable linker or a non-cleavable linker. The use of a cleavablelinker in a IFNgR2 binding molecule as contemplated herein facilitatesthe release of a therapeutic agent into the intracellular cytoplasm uponinternalization of the IFNgR2 binding molecule. A non-cleavable linkerwould allow release upon digestion of the IFNgR2 binding molecule of orit could be used with an agent that does not require release from theantibody (e.g., an imaging agent).

In some embodiments, where the IFNgR2 binding molecule is an IFNgR2 sdAbin stable association with an additional agent joined via a linker. Alinker is a covalent linkage between two elements of a IFNgR2 bindingmolecule (e.g., a hIFNgR2 binding VHH and PEG polymer). A linker can bea covalent bond, chemical linker or a peptide linker. Suitable linkersinclude “flexible linkers” which are generally of sufficient length topermit some movement between the IFNgR2 binding sdAb and the linkedagent(s). Examples of chemical linkers include aryl acetylene, ethyleneglycol oligomers containing 2-10 monomer units, diamines, diacids, aminoacids, or combinations thereof. In some embodiments, the linker is apeptide linker. Suitable peptide linkers can be readily selected and canbe of any suitable length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5,6, 7, 8, 9, 10, 10^(−20, 20)-30, 30-50 or more than 50 amino acids.Suitable peptide linkers are known in the art, and include, for example,peptide linkers containing flexible amino acid residues such as glycineand serine. Examples of flexible linkers include glycine polymers(G)_(n), glycine-serine polymers, glycine-alanine polymers,alanine-serine polymers, and other flexible linkers. Glycine andglycine-serine polymers are relatively unstructured, and therefore canserve as a neutral tether between components. Further examples offlexible linkers include glycine polymers (G)_(n), glycine-alaninepolymers, alanine-serine polymers, glycine-serine polymers. Glycine andglycine-serine polymers are relatively unstructured, and therefore mayserve as a neutral tether between components. A multimer (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of such linker sequencesmay be linked together to provide flexible linkers that may be used toconjugate a heterologous amino acid sequence to IFNgR2 binding sdAbsdisclosed herein. In some embodiments the linkers have the formula(GGGS)n (SEQ ID NO: 167), (GGGSG)n (SEQ ID NO: 168), or (GGSG)n (SEQ IDNO: 169), wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8,9 and 10.

Immunomodulatory Agents

In some embodiments, a IFNgR2 binding molecule of the present disclosureis operably linked to one or more an immunomodulatory agent(immunoconjugates). Immunomodulatory agents that may conjugated to thehIFNgR2 binding sdAb of the present disclosure include, but are notlimited to, inactivated virus particles, inactivated bacterial toxinssuch as toxoid from diphtheria, tetanus, cholera, or leukotoxinmolecules, inactivated bacteria and dendritic cells. Suchimmunoconjugates are useful in facilitating an immune response againstthe IFNgR2 or cells expressing the IFNgR2.

Flag Tags

In one embodiment, the present disclosure provides a IFNgR2 bindingmolecule operably linked to one or more an antigenic tags, such as aFLAG sequence. FLAG sequences are recognized by biotinylated, highlyspecific, anti-FLAG antibodies, as described herein (see e.g., Blanar etal. (1992) Science 256:1014 and LeClair, et al. (1992) PNAS-USA89:8145). In some embodiments, the IFNgR2 binding sdAb polypeptidefurther comprises a C-terminal c-myc epitope tag.

Chelating Peptides

In one embodiment, the present disclosure provides a IFNgR2 bindingmolecule operably linked to one or more transition metal chelatingpolypeptide sequences. The conjugation of the IFNgR2 binding molecule toa transition metal chelating domain facilitates purification immobilizedmetal affinity chromatography (IMAC) as described in Smith, et al. U.S.Pat. No. 4,569,794 issued Feb. 11, 1986. Examples of transition metalchelating polypeptides useful in the practice of the present IFNgR2binding molecule are described in Smith, et al. supra and Dobeli, et al.U.S. Pat. No. 5,320,663 issued May 10, 1995, the entire teachings ofwhich are hereby incorporated by reference. Particular transition metalchelating polypeptides useful in the practice of the present IFNgR2binding molecule are polypeptides comprising 3-6 contiguous histidineresidues (SEQ ID NO: 170) such as a six-histidine (His)6 peptide (SEQ IDNO: 165) and are frequently referred to in the art as “His-tags.” Inaddition to providing a purification “handle” for the recombinantproteins, such chelating peptides may also facilitate the delivery ofradioisotopes in substantial accordance with the teaching of Anderson,et al., U.S. Pat. No. 5,439,829 issued Aug. 8, 1995.

Carrier Molecules

In some embodiments the IFNgR2 binding sdAbs of the present disclosuremay be conjugated to one or more carrier molecules. Carrier moleculesare typically large, slowly metabolized macromolecules which provide forstabilization and/or extended duration of action in vivo to distinguishsuch molecules from conventional carrier molecules used in thepreparation of pharmaceutical formulations as described below. Examplesof in vivo carriers that may be incorporated into IFNgR2 bindingmolecules, but are not limited to: proteins (including but not limitedto human serum albumin); fatty acids (acylation); polysaccharides(including but not limited to (N- and O-linked) sugars, sepharose,agarose, cellulose, or cellulose); polypeptdies amino acid copolymers;acylation, or polysialylation, an polyethylene glycol (PEG) polymers.

Water Soluble Polymers

In some embodiments, the IFNgR2 binding sdAb is conjugated to one ormore water-soluble polymers. Examples of water soluble polymers usefulin the practice of the present IFNgR2 binding molecule includepolyethylene glycol (PEG), poly-propylene glycol (PPG), polysaccharides(polyvinylpyrrolidone, copolymers of ethylene glycol and propyleneglycol, poly(oxyethylated polyol), polyolefinic alcohol,polysaccharides, poly-alpha-hydroxy acid, polyvinyl alcohol (PVA),polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or acombination thereof.

Polyethylene Glycol

In one embodiment, the carrier molecule is a polyethylene glycol (“PEG”)polymer. Conjugation of PEG polymers to proteins (PEGylation) is awell-established method for the extension of serum half-life ofbiological agents. The PEGylated polypeptide may be further referred toas monopegylated, dipegylated, tripegylated (and so forth) to denote apolypeptide comprising one, two, three (or more) PEG moieties attachedto the polypeptide, respectively. In some embodiments, the PEG may becovalently attached directly to the sdAb (e.g., through a lysine sidechain, sulfhydryl group of a cysteine or N-terminal amine) or optionallyemploy a linker between the PEG and the sdAb. In some embodiments, aIFNgR2 binding molecule comprises more than one PEG molecules each ofwhich is attached to a different amino acid residue. In someembodiments, the sdAb may be modified by the incorporation ofnon-natural amino acids with non-naturally occurring amino acid sidechains to facilitate site specific PEGylation. In other embodiments,cysteine residues may be substituted at one or more positions within thesdAb to facilitate site-specific PEGylation via the cysteine sulfhydrylside chain.

In some instances, the IFNgR2 binding molecules of the presentdisclosure possess an N-terminal glutamine (“TQ”) residue. N-terminalglutamine residues have been observed to spontaneously cyclyize to formpyroglutamate (pE) at or near physiological conditions. (See e.g., Liu,et al (2011) J. Biol. Chem. 286(13): 11211-11217). In some embodiments,the formation of pyroglutamate complicates N-terminal PEG conjugationparticularly when aldehyde chemistry is used for N-terminal PEGylation.Consequently, when PEGylating the IFNgR2 binding molecules of thepresent disclosure, particularly when aldehyde chemistry is to beemployed, the IFNgR2 binding molecules possessing an amino acid atposition 1 (e.g., 1Q) are substituted at position 1 with an alternativeamino acid or are deleted at position 1 (e.g., des-TQ). In someembodiments, the IFNgR2 binding molecules of the present disclosurecomprise an amino acid substitution selected from the group Q1E and Q1D.

PEGs suitable for conjugation to a polypeptide sequence are generallysoluble in water at room temperature, and have the general formula

R(O—CH₂—CH₂)_(n)O—R,

where R is hydrogen or a protective group such as an alkyl or an alkanolgroup, and where n is an integer from 1 to 1000. When R is a protectivegroup, it generally has from 1 to 8 carbons. The PEG can be linear orbranched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs arecontemplated by the present disclosure.

A molecular weight of the PEG used in a IFNgR2 binding molecule is notrestricted to any particular range. The PEG component of a IFNgR2binding molecule can have a molecular mass greater than about 5 kDa,greater than about 10 kDa, greater than about 15 kDa, greater than about20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greaterthan about 50 kDa. In some embodiments, the molecular mass is from about5 kDa to about 10 kDa, from about 5 kDa to about 15 kDa, from about 5kDa to about 20 kDa, from about 10 kDa to about 15 kDa, from about 10kDa to about 20 kDa, from about 10 kDa to about 25 kDa or from about 10kDa to about 30 kDa. Linear or branched PEG molecules having molecularweights from about 2,000 to about 80,000 daltons, alternatively about2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000daltons, alternatively about 10,000 to about 50,000 daltons,alternatively about 20,000 to about 50,000 daltons, alternatively about30,000 to about 50,000 daltons, alternatively about 20,000 to about40,000 daltons, alternatively about 30,000 to about 40,000 daltons. Inone embodiment of the IFNgR2 binding molecule, the PEG is a 40 kDbranched PEG comprising two 20 kD arms.

The present disclosure also contemplates a IFNgR2 binding moleculecomprising more than one PEG moiety wherein the PEGs have differentsizes values, and thus the various different PEGs are present inspecific ratios. For example, in the preparation of a PEGylated IFNgR2binding molecule, some compositions comprise a mixture of mono-, di-,tri-, and quadra-PEGylated sdAb conjugates. In some compositions, thepercentage of mono-PEGylated species is 18-25%, the percentage ofdi-PEGylated species is 50-66%, the percentage of tri-pegylated speciesis 12-16%, and the percentage of quadra-pegylated species up to 5%. Suchcomplex compositions can be produced by reaction conditions andpurification methods known in the art. Chromatography may be used toresolve conjugate fractions, and a fraction is then identified whichcontains the conjugate having, for example, the desired number of PEGsattached, purified free from unmodified protein sequences and fromconjugates having other numbers of PEGs attached.

PEGylation most frequently occurs at the α-amino group at the N-terminusof the polypeptide, the epsilon amino group on the side chain of lysineresidues, and the imidazole group on the side chain of histidineresidues. Since most recombinant polypeptides possess a single alpha anda number of epsilon amino and imidazole groups, numerous positionalisomers can be generated depending on the linker chemistry.

Two widely used first generation activated monomethoxy PEGs (mPEGs) aresuccinimdyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992)Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG(BTC-PEG; see, e.g., Dolence, et al. U.S. Pat. No. 5,650,234), whichreact preferentially with lysine residues to form a carbamate linkagebut are also known to react with histidine and tyrosine residues. Use ofa PEG-aldehyde linker targets a single site on the N-terminus of apolypeptide through reductive amination.

The PEG can be bound to a IFNgR2 binding molecule of the presentdisclosure via a terminal reactive group (a “spacer”) which mediates abond between the free amino or carboxyl groups of one or more of thepolypeptide sequences and polyethylene glycol. The PEG having the spacerwhich can be bound to the free amino group includesN-hydroxysuccinylimide polyethylene glycol, which can be prepared byactivating succinic acid ester of polyethylene glycol withN-hydroxysuccinylimide.

In some embodiments, the PEGylation of the sdAb is facilitated by theincorporation of non-natural amino acids bearing unique side chains tofacilitate site specific PEGylation. The incorporation of non-naturalamino acids into polypeptides to provide functional moieties to achievesite specific PEGylation of such polypeptides is known in the art. Seee.g., Ptacin, et al., PCT International Application No.PCT/US2018/045257 filed Aug. 3, 2018 and published Feb. 7, 2019 asInternational Publication Number WO 2019/028419A1.

The PEG moiety of the of a PEGylated IFNgR2 binding molecule may belinear or branched. Branched PEG derivatives, “star-PEGs” andmulti-armed PEGs are contemplated by the present disclosure. Specificembodiments PEGs useful in the practice of the present disclosureinclude a 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOFAmerica Corporation, One North Broadway, White Plains, N.Y. 10601 USA),10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright®ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), a 20 kDalinear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF, a 20 kDa linearPEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS,Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), a 20 kDa 2-arm branchedPEG-aldehyde the 20 kDA PEG-aldehyde comprising two 10 kDA linear PEGmolecules (e.g., Sunbright® GL2-200AL3, NOF), a 20 kDa 2-arm branchedPEG-NHS ester the 20 kDA PEG-NHS ester comprising two 10 kDA linear PEGmolecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), a 40kDa 2-arm branched PEG-aldehyde the 40 kDA PEG-aldehyde comprising two20 kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3), a 40 kDa2-arm branched PEG-NHS ester the 40 kDA PEG-NHS ester comprising two 20kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright®GL2-400GS2, NOF), a linear 30 kDa PEG-aldehyde (e.g., Sunbright®ME-300AL) and a linear 30 kDa PEG-NHS ester.

Fc Fusions

In some embodiments, the carrier molecule is a Fc molecule or amonomeric subunit thereof. In some embodiments, the dimeric Fc moleculemay be engineered to possess a “knob-into-hole modification.” Theknob-into-hole modification is more fully described in Ridgway, et al.(1996) Protein Engineering 9(7):617-621 and U.S. Pat. No. 5,731,168,issued Mar. 24, 1998, U.S. Pat. No. 7,642,228, issued Jan. 5, 2010, U.S.Pat. No. 7,695,936, issued Apr. 13, 2010, and U.S. Pat. No. 8,216,805,issued Jul. 10, 2012. The knob-into-hole modification refers to amodification at the interface between two immunoglobulin heavy chains inthe CH3 domain, wherein: i) in a CH3 domain of a first heavy chain, anamino acid residue is replaced with an amino acid residue having alarger side chain (e.g., tyrosine or tryptophan) creating a projectionfrom the surface (“knob”) and ii) in the CH3 domain of a second heavychain, an amino acid residue is replaced with an amino acid residuehaving a smaller side chain (e.g., alanine or threonine), therebygenerating a cavity (“hole”) within at interface in the second CH3domain within which the protruding side chain of the first CH3 domain(“knob”) is received by the cavity in the second CH3 domain. In oneembodiment, the “knob-into-hole modification” comprises the amino acidsubstitution T366W and optionally the amino acid substitution S354C inone of the antibody heavy chains, and the amino acid substitutionsT366S, L368A, Y407V and optionally Y349C in the other one of theantibody heavy chains. Furthermore, the Fc domains may be modified bythe introduction of cysteine residues at positions S354 and Y349 whichresults in a stabilizing disulfide bridge between the two antibody heavychains in the Fe region (Carter, et al. (2001) Immunol Methods 248,7-15). The knob-into-hole format is used to facilitate the expression ofa first polypeptide (e.g., an IFNgR2 binding sdAb) on a first Fc monomerwith a “knob” modification and a second polypeptide on the second Fcmonomer possessing a “hole” modification to facilitate the expression ofheterodimeric polypeptide conjugates.

Targeting Domains

In some embodiments, the IFNgR2 binding molecule is provided as acomponent of a multivalent (e.g., bivalent) fusion protein with apolypeptide sequence (“targeting domain”) to facilitate selectivebinding to particular cell type or tissue expressing a cell surfacemolecule that specifically binds to such targeting domain, optionallyincorporating a linker between the IFNgR2 binding sdAb sequence and thesequence of the targeting domain of the fusion protein.

In some embodiments of the IFNgR2 binding molecule, the IFNgR2 bindingmolecule may be targeted to a particular cell type cell by incorporationof a targeting domain into the structure of the IFNgR2 bindingmolecules. As used herein, the term targeting domain refers to a moietythat specifically binds to a molecule expressed on the surface of atarget cell. The targeting domain may be any moiety that specificallybinds to one or more cell surface molecules (e.g., T cell receptor)expressed on the surface of a target cell. In some embodiments, thetarget cell is a T cell. In some embodiments, the target cell is aIFNgR2⁺ T cell.

In some embodiments, the targeting domain is a ligand for a receptor. Insome embodiments, the targeting domain is a ligand for a receptorexpressed on the surface of a T cell. In some embodiments, the ligand isa cytokine. In some embodiments, the cytokine includes but is notlimited to the group consisting interleukins, interferons, andfunctional derivatives thereof. In some embodiments, the cytokineincludes but is not limited to the group consisting IL2, IL3, IL4, IL7,IL9, IL12, IL15, IL18, IL21, IL22, IL23, IL27, IL28, IL34, and modifiedversions or fragments thereof that bind to their cognate ligandexpressed on the surface of a T-cell. In some embodiments, the cytokineincludes but is not limited to the group consisting of interferon alpha,interferon a2b, interferon gamma, or interferon lambda and modifiedversions or fragments thereof that bind to their cognate ligandexpressed on the surface of a T-cell.

In another aspect, the present disclosure provides a multivalent bindingmolecule, the multivalent binding molecule comprising: (a) a IFNgR2binding molecule and (b) a second binding molecule that specificallybinds to the extracellular domain of a second cell surface molecule,wherein the IFNgR2 binding molecule and second binding molecule areoperably linked, optionally through a chemical or polypeptide linker. Insome embodiments, the IFNgR2 binding molecules of the present disclosureare useful in the preparation of the multivalent binding moleculesdescribed in Gonzalez, et al. PCT/US2018/021301 published as WO2018/182935 A1 on Oct. 4, 2018. In accordance with the teaching ofGonzalez, the second binding molecule specifically binds to theextracellular domain of: (i) a component of cytokine receptor other thana receptor of which IFGgR2 forms a signaling complex in response to anatural ligand (e.g. IFNgR2) that activates the JAK/STAT pathway in thecell; (ii) a receptor tyrosine kinase; or (iii) a TNFR superfamilymember. In some embodiments, the second surface molecule is a tyrosinekinase selected from EGFR, ErbB2, ErbB3, ErbB4, InsR, IGF1R, InsRR,PDGFRα, PDGFRβ, CSF1R/Fms, cKit, Flt-3/Flk2, VEGFR1, VEGFR2, VEGFR3,FGFR1, FGFR2, FGFR3, FGFR4, PTK7/CCK4, TrkA, TrkB, TrkC, Ror1, Ror2,MuSK, Met, Ron, Axl, Mer, Tyro3, Tie1, Tie2, EphA1-8, EphA10, EphB1-4,EphB6, Ret, Ryk, DDR1, DDR2, Ros, LMR1, LMR2, LMR3, ALK, LTK,SuRTK106/STYK1. In some embodiments, the second surface molecule is aTNFR superfamily member is selected from TNFR1 (TNFRSFlA), TNFR2(TNFRSFlB; TNFRSF2), 41-BB (TNFRSF9); AITR (TNFRSF18); BCMA (TNFRSF17),CD27 (TNFRSF7), CD30 (TNFRSF8), CD40 (TNFRSF5), Death Receptor 1(TNFRSF10C), Death Receptor-3 (TNFRSF25), Death Receptor 4 (TNFRSF10A),Death Receptor 5 (TNFRSF10B), Death Receptor-6 (TNFRSF21), DecoyReceptor-3 (TNFRSF6B), Decoy Receptor 2 (TNFRSF10D), EDAR, Fas(TNFRSF6), HVEM (TNFRSF14), LTBR (TNFRSF3), OX40 (TNFRSF4), RANK(TNFRSF11A), TACI (TNFRSF13B), Troy (TNFRSF19), XEDAR (TNFRSF27),Osteoprotegerin (TNFRSF11B), TWEAK receptor (TNFRSF12A), BAFF Receptor(TNFRSF13C), NGF receptor (TNFRSF16).

In some embodiments, the targeting domain of the IFNgR2 binding moleculeis an antibody (as defined hereinabove to include molecules such asVHHs, scFvs, etc.) Examples of antibodies that may incorporated as atargeting domain of a IFNgR2 binding molecule include but are notlimited to the group consisting of: anti-GD2 antibodies, anti-BCMAantibodies, anti-CD19 antibodies, anti-CD33 antibodies, anti-CD38antibodies, anti-CD70 antibodies, anti-GD2 antibodies and IL3Ra2antibodies, anti-CD19 antibodies, anti-mesothelin antibodies, anti-Her2antibodies, anti-EpCam antibodies, anti-Muc antibodies, anti-ROR1antibodies, anti-CD133 antibodies, anti-CEA antibodies, anti-PSMAantibodies, anti-EGRFRVIII antibodies, anti-PSCA antibodies, anti-GPC3antibodies, anti-Pan-ErbB antibodies, and anti-FAP antibodies.

The antibody or antigen-binding fragment thereof can also be linked toanother antibody to form, e.g., a bispecific or a multispecificantibody.

Labels

In some embodiments, IFNgR2 binding molecules of the present disclosureare operably linked to a label. In some embodiments, the label isconjugated to the IFNgR2 binding molecule to facilitate use as imagingagent, diagnostic agent, or for use in cell sorting procedures. The termlabels includes but is not limited to fluorescent labels, a biologicallyactive enzyme labels, a radioisotopes (e.g., a radioactive ions), anuclear magnetic resonance active labels, a luminescent labels, or amagnetic compound. In one embodiment a IFNgR2 binding sdAb (e.g., aIFNgR2 binding VHH) molecule in stable association (e.g., covalent,coordinate covalent) with an imaging labels. The term imaging labels isused to describe any of a variety of compounds a signature thatfacilitates identification, tracing and/or localization of the IFNgR2binding sdAb (or its metabolites) using diagnostic procedures. Examplesof imaging labels include, but are not limited to, fluorescentcompounds, radioactive compounds, and compounds opaque to imagingmethods (e.g., X-ray, ultrasound). Examples of radioactive compoundsuseful as imaging label include but are not limited to Technetium-99m(^(99m)Tc), Indium-111(¹¹¹In), Iodine-131 (¹³¹I), Iodine-123(¹²³I),Iodine-125 (¹²⁵I), Gallium-67 (⁶⁷Ga), and Lutetium-177 (¹⁷⁷Lu),phosphorus (³²P), carbon (¹⁴C), tritium (³H), yttrium (⁹⁰Y), actinium(²²⁵Ac), astatine (²¹¹At), rhenium (¹⁸⁶ Re), bismuth (²¹²Bi or ²¹³Bi),and rhodium (¹⁸⁸Rh).

Therapeutic Agents

In some embodiments, IFNgR2 binding molecules of the present disclosureare operably linked to a therapeutic agent. Examples of therapeuticagents include therapeutic small molecule (e.g., chemotherapeuticagents) or biologic therapeutic agents including antibodies, cytoxic orcytostatic compounds, a radioisotope, molecules of plant, fungal, orbacterial origin, or biological proteins (e.g., protein toxins) orparticles (e.g., nano-particles or recombinant viral particles, e.g.,via a viral coat protein), therapeutic antibodies, chemotherapeuticagents, as described more fully herein.

In some embodiments, the therapeutic agent which may be incorporatedinto the IFNgR2 binding molecules of the present disclosure isshort-range radiation emitters, including, for example, short-range,high-energy a-emitters. Examples of such radioisotope include analpha-emitter, a beta-emitter, a gamma-emitter or a beta/gamma emitter.Radioisotopes useful as therapeutic agents include yttrium 90 (⁹⁰Y),lutetium-177 (¹⁷⁷Lu), actinium-225 (²²⁵Ac), astatine-211 (²¹¹At),rhenium-186 (¹⁸⁶Re), bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), andrhodium-188 (¹⁸⁸Rh) Synthesis of IFNgR2 binding molecules:

In some embodiments, the IFNgR2 binding molecules of the presentdisclosure are polypeptides. However, in some embodiments, only aportion of the IFNgR2 binding molecule is a polypeptide, for examplewhere the IFNgR2 binding molecule comprises a non-peptidyl domain (e.g.,a PEG IFNgR2 binding sdAb conjugate, a radionucleotide IFNgR2 bindingsdAb conjugate, or a small molecule IFNgR2 binding sdAb conjugate). Thefollowing provides guidance to enable the solid phase and recombinantsynthesis of the polypeptide portions (domains) of IFNgR2 bindingmolecules of the present disclosure. In those embodiments where only aportion of the IFNgR2 binding molecule is a polypeptide, it will beunderstood that the peptidyl domain(s) of the IFNgR2 binding moleculeare an intermediate in the process which may undergo further processingto complete the synthesis of the desired IFNgR2 binding molecules. Thepolypeptide domains of IFNgR2 binding molecules may be produced byconventional methodology for the construction of polypeptides includingrecombinant or solid phase syntheses as described in more detail below.

Chemical Synthesis

In addition to generating mutant polypeptides via expression of nucleicacid molecules that have been altered by recombinant molecularbiological techniques, polypeptide domains of IFNgR2 binding moleculescan be chemically synthesized. Chemically synthesized polypeptides areroutinely generated by those of skill in the art. Chemical synthesisincludes direct synthesis of a peptide by chemical means of thepolypeptide domains of IFNgR2 binding molecules exhibiting theproperties described. This method can incorporate both natural andunnatural amino acids at desired positions that facilitate linkage ofparticular molecules (e.g., PEG).

In some embodiments, the polypeptide domains of IFNgR2 binding moleculesof the present disclosure may be prepared by chemical synthesis. Thechemical synthesis of the polypeptide domains of IFNgR2 bindingmolecules may proceed via liquid-phase or solid-phase. Solid-phasepeptide synthesis (SPPS) allows the incorporation of unnatural aminoacids and/or peptide/protein backbone modification. Various forms ofSPPS are available for synthesizing the polypeptide domains of IFNgR2binding molecules of the present disclosure are known in the art (e.g.,Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero J. A. etal., (2005) Protein Pept Lett. 12:723-8). In the course of chemicalsynthesis, the alpha functions and any reactive side chains mayprotected with acid-labile or base-labile groups that are stable underthe conditions for linking amide bonds but can readily be cleavedwithout impairing the peptide chain that has formed.

In the solid phase synthesis, either the N-terminal or C-terminal aminoacid may be coupled to a suitable support material. Suitable supportmaterials are those which are inert towards the reagents and reactionconditions for the stepwise condensation and cleavage reactions of thesynthesis process and which do not dissolve in the reaction media beingused. Examples of commercially available support materials includestyrene/divinylbenzene copolymers which have been modified with reactivegroups and/or polyethylene glycol; chloromethylatedstyrene/divinylbenzene copolymers; hydroxymethylated or aminomethylatedstyrene/divinylbenzene copolymers; and the like. The successive couplingof the protected amino acids can be carried out according toconventional methods in peptide synthesis, typically in an automatedpeptide synthesizer.

At the end of the solid phase synthesis, the peptide is cleaved from thesupport material while simultaneously cleaving the side chain protectinggroups. The peptide obtained can be purified by various chromatographicmethods including but not limited to hydrophobic adsorptionchromatography, ion exchange chromatography, distributionchromatography, high pressure liquid chromatography (HPLC) andreversed-phase HPLC.

Recombinant Production

Alternatively, polypeptide domains of IFNgR2 binding molecules of thepresent disclosure may be produced by recombinant DNA technology. In thetypical practice of recombinant production of polypeptides, a nucleicacid sequence encoding the desired polypeptide is incorporated into anexpression vector suitable for the host cell in which expression will beaccomplish, the nucleic acid sequence being operably linked to one ormore expression control sequences encoding by the vector and functionalin the target host cell. The recombinant protein may be recoveredthrough disruption of the host cell or from the cell medium if asecretion leader sequence (signal peptide) is incorporated into thepolypeptide. The recombinant protein may be purified and concentratedfor further use including incorporation.

Synthesis of Nucleic Acid Sequences Encoding the IFNgR2 Binding Molecule

In some embodiments, the polypeptide domains of IFNgR2 binding moleculeis produced by recombinant methods using a nucleic acid sequenceencoding the polypeptide domains of IFNgR2 binding molecule (or fusionprotein comprising the polypeptide domains of IFNgR2 binding molecule).The nucleic acid sequence encoding the desired polypeptide domains ofIFNgR2 binding molecule can be synthesized by chemical means using anoligonucleotide synthesizer.

The nucleic acid molecules are not limited to sequences that encodepolypeptides; some or all of the non-coding sequences that lie upstreamor downstream from a coding sequence (e.g., the coding sequence of thepolypeptide domains of IFNgR2 binding molecule) can also be included.Those of ordinary skill in the art of molecular biology are familiarwith routine procedures for isolating nucleic acid molecules. They can,for example, be generated by treatment of genomic DNA with restrictionendonucleases, or by performance of the polymerase chain reaction (PCR).In the event the nucleic acid molecule is a ribonucleic acid (RNA),molecules can be produced, for example, by in vitro transcription.

The nucleic acid molecules encoding the polypeptide domains of IFNgR2binding molecule (and fusions thereof) may contain naturally occurringsequences or sequences that differ from those that occur naturally, but,due to the degeneracy of the genetic code, encode the same polypeptide.These nucleic acid molecules can consist of RNA or DNA (for example,genomic DNA, cDNA, or synthetic DNA, such as that produced byphosphoramidite-based synthesis), or combinations or modifications ofthe nucleotides within these types of nucleic acids. In addition, thenucleic acid molecules can be double-stranded or single-stranded (i.e.,either a sense or an antisense strand).

Nucleic acid sequences encoding the polypeptide domains of the IFNgR2binding molecule may be obtained from various commercial sources thatprovide custom synthesis of nucleic acid sequences. Amino acid sequencevariants of the HUMAN IFNgR2 binding molecules of the present disclosureare prepared by introducing appropriate nucleotide changes into thecoding sequence based on the genetic code which is well known in theart. Such variants represent insertions, substitutions, and/or specifieddeletions of, residues as noted. Any combination of insertion,substitution, and/or specified deletion can be made to arrive at thefinal construct, provided that the final construct possesses the desiredbiological activity as defined herein.

Methods for constructing a DNA sequence encoding the polypeptide domainsof IFNgR2 binding molecule and expressing those sequences in a suitablytransformed host include, but are not limited to, using a PCR-assistedmutagenesis technique. Mutations that consist of deletions or additionsof amino acid residues to polypeptide domains of IFNgR2 binding moleculecan also be made with standard recombinant techniques. In the event of adeletion or addition, the nucleic acid molecule encoding polypeptidedomains of IFNgR2 binding molecule is optionally digested with anappropriate restriction endonuclease. The resulting fragment can eitherbe expressed directly or manipulated further by, for example, ligatingit to a second fragment. The ligation may be facilitated if the two endsof the nucleic acid molecules contain complementary nucleotides thatoverlap one another, but blunt-ended fragments can also be ligated.PCR-generated nucleic acids can also be used to generate various mutantsequences.

A polypeptide domain of IFNgR2 binding molecules of the presentdisclosure may be produced recombinantly not only directly, but also asa fusion polypeptide with a heterologous polypeptide, e.g., a signalsequence or other polypeptide having a specific cleavage site at theN-terminus or C-terminus of the mature IFNgR2 binding molecule. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the coding sequence that is inserted into the vector. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In some embodiments, the signal sequence is the signalsequence that is natively associated with the IFNgR2 binding molecule(i.e. the human IFNgR2 signal sequence). The inclusion of a signalsequence depends on whether it is desired to secrete the IFNgR2 bindingmolecule from the recombinant cells in which it is made. If the chosencells are prokaryotic, it generally is preferred that the DNA sequencenot encode a signal sequence. If the chosen cells are eukaryotic, itgenerally is preferred that a signal sequence be encoded and mostpreferably that the wild type IL-2 signal sequence be used.Alternatively, heterologous mammalian signal sequences may be suitable,such as signal sequences from secreted polypeptides of the same orrelated species, as well as viral secretory leaders, for example, theherpes simplex gD signal. When the recombinant host cell is a yeast cellsuch as Saccharomyces cerevisiae, the alpha mating factor secretionsignal sequence may be employed to achieve extracellular secretion ofthe IFNgR2 binding molecule into the culture medium as described inSingh, U.S. Pat. No. 7,198,919 B1.

In the event the polypeptide domain of IFNgR2 binding molecules to beexpressed is to be expressed as a chimera (e.g., a fusion proteincomprising a IFNgR2 binding molecule and a heterologous polypeptidesequence), the chimeric protein can be encoded by a hybrid nucleic acidmolecule comprising a first sequence that encodes all or part of thepolypeptide domains of IFNgR2 binding molecule and a second sequencethat encodes all or part of the heterologous polypeptide. For example,polypeptide domains of IFNgR2 binding molecules described herein may befused to a hexa-histidine tag (SEQ ID NO: 165) to facilitatepurification of bacterially expressed protein, or to a hemagglutinin tagto facilitate purification of protein expressed in eukaryotic cells. Byfirst and second, it should not be understood as limiting to theorientation of the elements of the fusion protein and a heterologouspolypeptide can be linked at either the N-terminus and/or C-terminus ofthe polypeptide domains of IFNgR2 binding molecule. For example, theN-terminus may be linked to a targeting domain and the C-terminus linkedto a hexa-histidine tag (SEQ ID NO: 165) purification handle.

The complete amino acid sequence of the polypeptide domain of IFNgR2binding molecule (or fusion/chimera) to be expressed can be used toconstruct a back-translated gene. A DNA oligomer containing a nucleotidesequence coding for the polypeptide domain of IFNgR2 binding moleculescan be synthesized. For example, several small oligonucleotides codingfor portions of the desired polypeptide can be synthesized and thenligated. The individual oligonucleotides typically contain 5′ or 3′overhangs for complementary assembly.

In some embodiments, the nucleic acid sequence encoding the polypeptidedomain of the IFNgR2 binding molecule may be “codon optimized” tofacilitate expression in a particular host cell type. Techniques forcodon optimization in a wide variety of expression systems, includingmammalian, yeast and bacterial host cells, are well known in the andthere are online tools to provide for a codon optimized sequences forexpression in a variety of host cell types. See e.g., Hawash, et al.,(2017) 9:46-53 and Mauro and Chappell in Recombinant Protein Expressionin Mammalian Cells: Methods and Protocols, edited by David Hacker (HumanPress New York). Additionally, there are a variety of web based on-linesoftware packages that are freely available to assist in the preparationof codon optimized nucleic acid sequences.

Expression Vectors

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the nucleic acid sequence encoding polypeptide domains ofIFNgR2 binding molecule will be inserted into an expression vector. Avariety of expression vectors for uses in various host cells areavailable and are typically selected based on the host cell forexpression. An expression vector typically includes, but is not limitedto, one or more of the following: an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Vectors include viral vectors, plasmid vectors,integrating vectors, and the like. Plasmids are examples of non-viralvectors. To facilitate efficient expression of the recombinantpolypeptide, the nucleic acid sequence encoding the polypeptide sequenceto be expressed is operably linked to transcriptional and translationalregulatory control sequences that are functional in the chosenexpression host.

Expression vectors typically contain a selection gene, also termed aselectable marker. This gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media.

Expression vectors for polypeptide domain of IFNgR2 binding molecules ofthe present disclosure contain a regulatory sequence that is recognizedby the host organism and is operably linked to nucleic acid sequenceencoding the polypeptide domains of IFNgR2 binding molecule. The terms“regulatory control sequence,” “regulatory sequence” or “expressioncontrol sequence” are used interchangeably herein to refer to promoters,enhancers, and other expression control elements (e.g., polyadenylationsignals). See, for example, Goeddel (1990) in Gene ExpressionTechnology: Methods in Enzymology 185 (Academic Press, San Diego Calif.USA Regulatory sequences include those that direct constitute expressionof a nucleotide sequence in many types of host cells and those thatdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. In selectingan expression control sequence, a variety of factors understood by oneof skill in the art are to be considered. These include, for example,the relative strength of the sequence, its controllability, and itscompatibility with the actual DNA sequence encoding the subject IFNgR2binding molecule, particularly as regards potential secondarystructures.

In some embodiments, the regulatory sequence is a promoter, which isselected based on, for example, the cell type in which expression issought. Promoters are untranslated sequences located upstream (5′) tothe start codon of a structural gene (generally within about 100 to 1000bp) that control the transcription and translation of particular nucleicacid sequence to which they are operably linked. Such promoterstypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g., the presence or absence of a nutrient or a change intemperature. A large number of promoters recognized by a variety ofpotential host cells are well known.

A T7 promoter can be used in bacteria, a polyhedrin promoter can be usedin insect cells, and a cytomegalovirus or metallothionein promoter canbe used in mammalian cells. Also, in the case of higher eukaryotes,tissue-specific and cell type-specific promoters are widely available.These promoters are so named for their ability to direct expression of anucleic acid molecule in a given tissue or cell type within the body.Skilled artisans are well aware of numerous promoters and otherregulatory elements which can be used to direct expression of nucleicacids.

Transcription from vectors in mammalian host cells may be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as human adenovirusserotype 5), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus (such as murine stem cell virus),hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter, PGK(phosphoglycerate kinase), or an immunoglobulin promoter, fromheat-shock promoters, provided such promoters are compatible with thehost cell systems. The early and late promoters of the SV40 virus areconveniently obtained as an SV40 restriction fragment that also containsthe SV40 viral origin of replication.

Transcription by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, which act on a promoter toincrease its transcription. Enhancers are relatively orientation andposition independent, having been found 5′ and 3′ to the transcriptionunit, within an intron, as well as within the coding sequence itself.Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, alpha-fetoprotein, and insulin). Typically, however,one will use an enhancer from a eukaryotic cell virus. Examples includethe SV40 enhancer on the late side of the replication origin, thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the expression vector at a position 5′ or3′ to the coding sequence but is preferably located at a site 5′ fromthe promoter. Expression vectors used in eukaryotic host cells will alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. Construction of suitable vectors containing one or more of theabove-listed components employs standard techniques.

In addition to sequences that facilitate transcription of the insertednucleic acid molecule, vectors can contain origins of replication, andother genes that encode a selectable marker. For example, theneomycin-resistance (neoR) gene imparts G418 resistance to cells inwhich it is expressed, and thus permits phenotypic selection of thetransfected cells. Additional examples of marker or reporter genesinclude beta-lactamase, chloramphenicol acetyltransferase (CAT),adenosine deaminase (ADA), dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ(encoding beta-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). Those of skill in the art can readilydetermine whether a given regulatory element or selectable marker issuitable for use in a particular experimental context. Proper assemblyof the expression vector can be confirmed by nucleotide sequencing,restriction mapping, and expression of a biologically active polypeptidein a suitable host.

Host Cells

The present disclosure further provides prokaryotic or eukaryotic cellsthat contain and express a nucleic acid molecule that encodes apolypeptide domains of IFNgR2 binding molecule. A cell of the presentdisclosure is a transfected cell, i.e., a cell into which a nucleic acidmolecule, for example a nucleic acid molecule encoding a polypeptidedomains of IFNgR2 binding molecule, has been introduced by means ofrecombinant DNA techniques. The progeny of such a cell are alsoconsidered within the scope of the present disclosure.

Host cells are typically selected in accordance with their compatibilitywith the chosen expression vector, the toxicity of the product coded forby the DNA sequences of this IFNgR2 binding molecule, their secretioncharacteristics, their ability to fold the polypeptides correctly, theirfermentation or culture requirements, and the ease of purification ofthe products coded for by the DNA sequences. Suitable host cells forcloning or expressing the DNA in the vectors herein are the prokaryote,yeast, or higher eukaryote cells.

In some embodiments the recombinant polypeptide domains of IFNgR2binding molecule or biologically active variants thereof can also bemade in eukaryotes, such as yeast or human cells. Suitable eukaryotichost cells include insect cells (examples of Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39)); yeast cells (examples of vectors for expression in yeast S.cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz etal. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.)); ormammalian cells (mammalian expression vectors include pCDM8 (Seed (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)).

Examples of useful mammalian host cell lines are mouse L cells(L-M[TK-], ATCC #CRL-2648), monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or HEK293cells subcloned for growth in suspension culture; baby hamster kidneycells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mousesertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); Africangreen monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervicalcarcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2). In mammalian cells, the expressionvector's control functions are often provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus, and Simian Virus 40.

The polypeptide domains of IFNgR2 binding molecule can be produced in aprokaryotic host, such as the bacterium E. coli, or in a eukaryotichost, such as an insect cell (e.g., an Sf21 cell), or mammalian cells(e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells areavailable from many sources, including the American Type CultureCollection (Manassas, Va.). Artisans or ordinary skill are able to makesuch a determination. Furthermore, if guidance is required in selectingan expression system, skilled artisans may consult Ausubel et al.(Current Protocols in Molecular Biology, John Wiley and Sons, New York,N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual,1985 Suppl. 1987).

In some embodiments, the recombinant polypeptide domains of IFNgR2binding molecule may be glycosylated or unglycosylated depending on thehost organism used to produce the IFNgR2 binding molecule. If bacteriaare chosen as the host then the polypeptide domains of IFNgR2 bindingmolecule produced will be aglycosylated. Eukaryotic cells, on the otherhand, will glycosylate the recombinant polypeptide domains of IFNgR2binding molecule.

For other additional expression systems for both prokaryotic andeukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.).

Transfection

The expression constructs can be introduced into host cells to therebyproduce the recombinant polypeptide domains of IFNgR2 binding moleculedisclosed herein or to produce biologically active muteins thereof.Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. Suitable methodsfor transforming or transfecting host cells can be found in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.) and other standard molecularbiology laboratory manuals.

In order to facilitate transfection of the target cells, the target cellmay be exposed directly with the non-viral vector may under conditionsthat facilitate uptake of the non-viral vector. Examples of conditionswhich facilitate uptake of foreign nucleic acid by mammalian cells arewell known in the art and include but are not limited to chemical means(such as Lipofectamine®, Thermo-Fisher Scientific), high salt, andmagnetic fields (electroporation).

Cell Culture

Cells may be cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. Mammalian hostcells may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics, trace elements, and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression and will beapparent to the ordinarily skilled artisan.

Recovery of Recombinant Proteins

Recombinantly-produced IFNgR2 binding polypeptides can be recovered fromthe culture medium as a secreted polypeptide if a secretion leadersequence is employed. Alternatively, the IFNgR2 binding polypeptides canalso be recovered from host cell lysates. A protease inhibitor, such asphenyl methyl sulfonyl fluoride (PMSF) may be employed during therecovery phase from cell lysates to inhibit proteolytic degradationduring purification, and antibiotics may be included to prevent thegrowth of adventitious contaminants.

Purification

Various purification steps are known in the art and find use, e.g.,affinity chromatography. Affinity chromatography makes use of the highlyspecific binding sites usually present in biological macromolecules,separating molecules on their ability to bind a particular ligand.Covalent bonds attach the ligand to an insoluble, porous support mediumin a manner that overtly presents the ligand to the protein sample,thereby using natural specific binding of one molecular species toseparate and purify a second species from a mixture. Antibodies arecommonly used in affinity chromatography. Size selection steps may alsobe used, e.g., gel filtration chromatography (also known assize-exclusion chromatography or molecular sieve chromatography) is usedto separate proteins according to their size. In gel filtration, aprotein solution is passed through a column that is packed withsemipermeable porous resin. The semipermeable resin has a range of poresizes that determines the size of proteins that can be separated withthe column.

The recombinant polypeptide domains of IFNgR2 binding molecule producedby the transformed host can be purified according to any suitablemethod. IFNgR2 binding molecules can be isolated from inclusion bodiesgenerated in E. coli, or from conditioned medium from either mammalianor yeast cultures producing a given IFNgR2 binding molecule sing cationexchange, gel filtration, and or reverse phase liquid chromatography.

The substantially purified forms of the recombinant polypeptides can beused, e.g., as therapeutic agents, as described herein.

The biological activity of the recombinant polypeptide domains of IFNgR2binding molecule produced in accordance with the foregoing can beconfirmed by a IFNgR2 binding using procedures well known in the artincluding but not limited to competition ELISA, radioactive ligandbinding assays (e.g., saturation binding, Scatchard plot, nonlinearcurve fitting programs and competition binding assays); non-radioactiveligand binding assays (e.g., fluorescence polarization (FP),fluorescence resonance energy transfer (FRET) and surface plasmonresonance assays (see, e.g., Drescher et al., Methods Mol Biol493:323-343 (2009) with instrumentation commercially available from GEHealthcare Bio-Sciences such as the Biacore 8+, Biacore 5200, BiacoreT200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA01752)); liquid phase ligand binding assays (e.g., real-time polymerasechain reaction (RT-qPCR), and immunoprecipitation); and solid phaseligand binding assays (e.g., multiwell plate assays, on-bead ligandbinding assays, on-column ligand binding assays, and filter assays).

Methods of Use

Inhibition of IFNgR2 Receptor Activity

In one embodiment, the present disclosure provides a method ofmodulating the activity of cells expressing the IFNgR2 by theadministration of a composition comprising IFNgR2 binding molecule to asubject in an amount sufficient to interfere with the activity ofreceptors comprising the of IFNgR2. The present disclosure furtherprovides a method of modulating the activity of cells expressing theIFNgR2 in a mixed population of cells comprising contacting saidpopulation of cells, in vivo and/or ex vivo, with a IFNgR2 bindingmolecule or complex of the present disclosure to in an amount sufficientto interfere with the activity of receptors comprising the IFNgR2.

Isolation, Enrichment or Depletion of IFNgR2+ Cells From a BiologicalSample

In one embodiment, the present disclosure provides a method of use ofthe IFNgR2 binding molecules of the present disclosure useful in aprocess for in the isolation, enrichment or depletion of IFNgR2+ cellsfrom a biological sample comprising IFNgR2+ cells. The biological samplemay comprise cells of blood origin such as PBMC, T cells, B cells ofcell culture origin or of tissue origin such as brain or bone marrow.Processes suitable for the isolation, enrichment or depletion of IFNgR2+cells comprise centrifugation, filtration, magnetic cell sorting andfluorescent cell sorting by techniques well known in the art. Thepresent disclosure further provides a method for the treatment of asubject suffering from a disease, disorder or condition by theadministration of a therapeutically effective amount of a cell productenriched or depleted of IFNgR2+ cells through the use of a IFNgR2binding molecule as described herein.

In one embodiment, the sorting procedure employs a IFNgR2 bindingmolecule comprising a fluorescent label for use in FACS isolation ordepletion of IFNgR2+ cells from a sample. The fluorescent label may beattached to the sdAb of the IFNgR2 binding molecule directly (e.g., bychemical conjugation optionally employing a linker) or indirectly (e.g.,by biotinylation of the sdAb and binding of the biotinylated antibody toa streptavidin fluorochrome conjugate). Such fluorescently labelledIFNgR2+ cells may be separated from a mixed cell population usingconventional FACS technology.

In an alternative embodiment, the selection procedure employs IFNgR2binding molecules of the present disclosure (e.g., a IFNgR2 binding VHH)conjugated to magnetic particles which provide magnetic labeling of theIFNgR2+ cells for use in magnetic cell separation procedures. In oneembodiment the method comprises: (a) conjugation of one or more IFNgR2binding molecule of the present disclosure (e.g., a IFNgR2 binding VHH)to a magnetic particle; (b) creating a mixture by contacting thebiological sample with a quantity of the magnetic particles conjugatedto IFNgR2 binding molecule; (c) subjecting to a magnetic field such thatthe magnetically labelled IFNgR2+ cells are retained; (d) removing thenon-magnetically labelled cells from the mixture; and (e) removal of themagnetic field enabling isolation of the IFNgR2+ cells.

The cell selection procedure (e.g., FACS or magnetic separation) resultsin two products: (a) a population of cells depleted of IFNgR2+ cells and(b) a population of cells enriched for IFNgR2+ cells. Each of thesepopulations may be further processed by convention procedures toidentify particular IFNgR2+ or IFNgR2− cell subsets which may be usefulin research, diagnostic or clinical applications. For example, isolationof specific IFNgR2⁺ T cell subsets that also express one or more of CD4,CD8, CD19, CD25, and CD62L, further iterations of the using one or moreantibodies that specifically bind to CD4, CD8, CD19, CD25, and CD62Lantigens respectively by FACS or magnetic field separation by techniqueswell known in the art.

In one embodiment of the IFNgR2 binding molecule a humanized antibody orfragment thereof as disclosed herein may be used for depletion ofIFNgR2-expressing cells from a biological sample comprisingIFNgR2-expressing cells such peripheral blood or lymphoid tissue whichmay optionally be further processed for further isolation ofIFNgR2+naïve T cell subsets, isolation human IFNgR2+ memory T cells froma population of CD4+ or CD8+ cells, or isolation of human IFNgR2RA+naïveT cells from presorted CD4+ or CD8+ cells by depletion of IFNgR2+ cells.In one embodiment, the IFNgR2 binding molecule provides a method ofgenerating a population of cells enriched for naïve Tregs from abiological sample, the method comprising depleting IFNgR2+ cells using aIFNgR2 binding molecule of the present disclosure as described above,optionally further comprising the steps of depleting CD8+ and/or CD19+cells. The IFNgR2+ depleted cell population may optionally be furtherexpanded in vitro for particular cell types to in the preparation of acell product comprising a therapeutically effective amount of theIFNgR2+ depleted cell product which may be administered to a subjectsuffering from a disease, disorder or condition.

The IFNgR2+ enriched cell population may optionally be further expandedin vitro to in the preparation of a cell product comprising atherapeutically effective amount of the IFNgR2+ cells.

Inflammatory and Autoimmune Disorders

Disorders amenable to treatment with an IFNgR2 binding molecule(including pharmaceutically acceptable formulations comprising an IFNgR2binding molecules and/or the nucleic acid molecules that encode themincluding recombinant viruses encoding such an IFNgR2 binding molecules)of the present disclosure include inflammatory or autoimmune diseasesincluding but not limited to, organ rejection, graft versus hostdisease, autoimmune thyroid disease, multiple sclerosis, allergy,asthma, neurodegenerative diseases including Alzheimer's disease,systemic lupus erythramatosis (SLE), autoinflammatory diseases,inflammatory bowel disease (IBD), Crohn's disease, diabetes includingType 1 or type 2 diabetes, inflammation, autoimmune disease, atopicdiseases, paraneoplastic autoimmune diseases, cartilage inflammation,arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoidarthritis, juvenile rheumatoid arthritis, polyarticular juvenilerheumatoid arthritis, systemic onset juvenile rheumatoid arthritis,juvenile ankylosing spondylitis, juvenile enteropathic arthritis,juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome(Seronegativity Enthesopathy Arthropathy Syndrome), juveniledermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma,juvenile systemic lupus erythematosus, juvenile vasculitis,pauciarticular rheumatoidarthritis, polyarticular rheumatoidarthritis,systemic onset rheumatoidarthritis, ankylosing spondylitis, enteropathicarthritis, reactive arthritis, Reiter's syndrome, SEA Syndrome(Seronegativity, Enthesopathy, Arthropathy Syndrome).

Other examples of proliferative and/or differentiative disordersamenable to treatment with IFNgR2 binding molecules (includingpharmaceutically acceptable formulations comprising IFNgR2 bindingmolecules and/or the nucleic acid molecules that encode them includingrecombinant viruses encoding such IFNgR2 binding molecules) of thepresent disclosure include, but are not limited to, skin disorders. Theskin disorder may involve the aberrant activity of a cell or a group ofcells or layers in the dermal, epidermal, or hypodermal layer, or anabnormality in the dermal-epidermal junction. For example, the skindisorder may involve aberrant activity of keratinocytes (e.g.,hyperproliferative basal and immediately suprabasal keratinocytes),melanocytes, Langerhans cells, Merkel cells, immune cell, and othercells found in one or more of the epidermal layers, e.g., the stratumbasale (stratum germinativum), stratum spinosum, stratum granulosum,stratum lucidum or stratum corneum. In other embodiments, the disordermay involve aberrant activity of a dermal cell, for example, a dermalendothelial, fibroblast, immune cell (e.g., mast cell or macrophage)found in a dermal layer, for example, the papillary layer or thereticular layer.

Examples of inflammatory or autoimmune skin disorders include psoriasis,psoriatic arthritis, dermatitis (eczema), for example, exfoliativedermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasisrosacea, parapsoriasis, pityriasis lichenoiders, lichen planus, lichennitidus, ichthyosiform dermatosis, keratodermas, dermatosis, alopeciaareata, pyoderma gangrenosum, vitiligo, pemphigoid (e.g., ocularcicatricial pemphigoid or bullous pemphigoid), urticaria, prokeratosis,rheumatoid arthritis that involves hyperproliferation and inflammationof epithelial-related cells lining the joint capsule; dermatitises suchas seborrheic dermatitis and solar dermatitis; keratoses such asseborrheic keratosis, senile keratosis, actinic keratosis, photo-inducedkeratosis, and keratosis follicularis; acne vulgaris; keloids andprophylaxis against keloid formation; nevi; warts including verruca,condyloma or condyloma acuminatum, and human papilloma viral (HPV)infections such as venereal warts; leukoplakia; lichen planus; andkeratitis. The skin disorder can be dermatitis, e.g., atopic dermatitisor allergic dermatitis, or psoriasis.

The compositions of the present disclosure (including pharmaceuticallyacceptable formulations comprising IFNgR2 binding molecules and/or thenucleic acid molecules that encode them including recombinant virusesencoding such IFNgR2 binding molecules) can also be administered to apatient who is suffering from (or may suffer from) psoriasis orpsoriatic disorders. The term “psoriasis” is intended to have itsmedical meaning, namely, a disease which afflicts primarily the skin andproduces raised, thickened, scaling, nonscarring lesions. The lesionsare usually sharply demarcated erythematous papules covered withoverlapping shiny scales. The scales are typically silvery or slightlyopalescent. Involvement of the nails frequently occurs resulting inpitting, separation of the nail, thickening and discoloration. Psoriasisis sometimes associated with arthritis, and it may be crippling.Hyperproliferation of keratinocytes is a key feature of psoriaticepidermal hyperplasia along with epidermal inflammation and reduceddifferentiation of keratinocytes. Multiple mechanisms have been invokedto explain the keratinocyte hyperproliferation that characterizespsoriasis. Disordered cellular immunity has also been implicated in thepathogenesis of psoriasis. Examples of psoriatic disorders includechronic stationary psoriasis, plaque psoriasis, moderate to severeplaque psoriasis, psoriasis vulgaris, eruptive psoriasis, psoriaticerythroderma, generalized pustular psoriasis, annular pustularpsoriasis, or localized pustular psoriasis.

Combination with Supplementary Therapeutic Agents

The present disclosure provides for the use of the IFNgR2 bindingmolecules of the present disclosure in combination with one or moreadditional active agents (“supplementary agents”). Such furthercombinations are referred to interchangeably as “supplementarycombinations” or “supplementary combination therapy” and thosetherapeutic agents that are used in combination with IFNgR2 bindingmolecules of the present disclosure are referred to as “supplementaryagents.” As used herein, the term “supplementary agents” includes agentsthat can be administered or introduced separately, for example,formulated separately for separate administration (e.g., as may beprovided in a kit) and/or therapies that can be administered orintroduced in combination with the IFNgR2 binding molecules.

As used herein, the term “in combination with” when used in reference tothe administration of multiple agents to a subject refers to theadministration of a first agent at least one additional (i.e. second,third, fourth, fifth, etc.) agent to a subject. For purposes of thepresent invention, one agent (e.g., IFNgR2 binding molecule) isconsidered to be administered in combination with a second agent (e.g.,a modulator of an immune checkpoint pathway) if the biological effectresulting from the administration of the first agent persists in thesubject at the time of administration of the second agent such that thetherapeutic effects of the first agent and second agent overlap. Forexample, the PD1 immune checkpoint inhibitors (e.g., nivolumab orpembrolizumab) are typically administered by IV infusion every two weeksor every three weeks while the IFNgR2 binding molecules of the presentdisclosure are typically administered more frequently, e.g., daily, BID,or weekly. However, the administration of the first agent (e.g.,pembrolizumab) provides a therapeutic effect over an extended time andthe administration of the second agent (e.g., an IFNgR2 bindingmolecule) provides its therapeutic effect while the therapeutic effectof the first agent remains ongoing such that the second agent isconsidered to be administered in combination with the first agent, eventhough the first agent may have been administered at a point in timesignificantly distant (e.g., days or weeks) from the time ofadministration of the second agent. In one embodiment, one agent isconsidered to be administered in combination with a second agent if thefirst and second agents are administered simultaneously (within 30minutes of each other), contemporaneously or sequentially. In someembodiments, a first agent is deemed to be administered“contemporaneously” with a second agent if first and second agents areadministered within about 24 hours of each another, preferably withinabout 12 hours of each other, preferably within about 6 hours of eachother, preferably within about 2 hours of each other, or preferablywithin about 30 minutes of each other. The term “in combination with”shall also understood to apply to the situation where a first agent anda second agent are co-formulated in single pharmaceutically acceptableformulation and the co-formulation is administered to a subject. Incertain embodiments, the IFNgR2 binding molecule and the supplementaryagent(s) are administered or applied sequentially, e.g., where one agentis administered prior to one or more other agents. In other embodiments,the IFNgR2 binding molecule and the supplementary agent(s) areadministered simultaneously, e.g., where two or more agents areadministered at or about the same time; the two or more agents may bepresent in two or more separate formulations or combined into a singleformulation (i.e., a co-formulation). Regardless of whether the agentsare administered sequentially or simultaneously, they are considered tobe administered in combination for purposes of the present disclosure.

Supplemental Agents Useful in the Treatment of Inflammatory orAutoimmune Disorders

In some embodiments, the method further comprises administering of theIFNgR2 binding molecule of the present disclosure in combination withone or more supplementary agents selected from the group consisting of acorticosteroid, a Janus kinase inhibitor, a calcineurin inhibitor, amTor inhibitor, an IMDH inhibitor, a biologic, a vaccine, and atherapeutic antibody. In certain embodiments, the therapeutic antibodyis an antibody that binds a protein selected from the group consistingof BLyS, CD11a, CD20, CD25, CD3, CD52, IgEIL12/IL23, IL17a, ILTB, IL4Rα,IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A, and VLA-4.

In some embodiments, the supplementary agent is one or more agentsselected from the group consisting of corticosteroids (including but notlimited to prednisone, budesonide, prednilisone), Janus kinaseinhibitors (including but not limited to tofacitinib (Xeljanz®),calcineurin inhibitors (including but not limited to cyclosporine andtacrolimus), mTor inhibitors (including but not limited to sirolimus andeverolimus), IMDH inhibitors (including but not limited to azathioprine,leflunomide and mycophenolate), biologics such as abatcept (Orencia®) oretanercept (Enbrel®), and therapeutic antibodies.

Examples of therapeutic antibodies that may be administered assupplementary agents in combination with the IFNgR2 binding molecules ofthe present disclosure in the treatment of autoimmune disease includebut are not limited to anti-CD25 antibodies (e.g. daclizumab andbasiliximab), anti-VLA-4 antibodies (e.g. natalizumab), anti-CD52antibodies (e.g. alemtuzumab), anti-CD20 antibodies (e.g. rituximab,ocrelizumab), anti-TNF antibodies (e.g. infliximab, and adalimumab),anti-IL6R antibodies (e.g. tocilizumab), anti-TNFα antibodies (e.g.adalimumab (Humira®), golimumab, and infliximab), anti-integrin-α4β7antibodies (e.g. vedolizumab), anti-IL17a antibodies (e.g. brodalumab orsecukinumab), anti-IL4Rα antibodies (e.g. dupilumab), anti-RANKLantibodies, IL6R antibodies, anti-IL1B antibodies (e.g. canakinumab),anti-CD1 Ta antibodies (e.g. efalizumab), anti-CD3 antibodies (e.g.muramonab), anti-IL5 antibodies (e.g. mepolizumab, reslizumab),anti-BLyS antibodies (e.g. belimumab); and anti-IL12/IL23 antibodies(e.g ustekinumab).

Many therapeutic antibodies have been approved for clinical use againstautoimmune disease. Examples of antibodies approved by the United StatesFood and Drug Administration (FDA) for use in the treatment ofautoimmune diseases in a subject suffering therefrom that may beadministered as supplementary agents in combination with the IFNgR2binding molecules of the present disclosure (and optionally additionalsupplementary agents) for the treatment of the indicated autoimmunedisease are provided in Table 4 below:

TABLE 4 Antibodies Useful as Supplmentary Agents In the Treatment ofAutoimmune and Inflammatory Disease Name Target Therapeutic Indicationefalizumab CD11a Psoriasis belimumab BLyS Systemic lupus erythematosusocrelizumab CD20 Multiple sclerosis rituximab CD20 Multiple sclerosisbasiliximab CD25 Transplantation rejection daclizumab CD25Transplantation rejection muromonab CD3 Transplantation rejectionalemtuzumab CD52 Multiple sclerosis omalizumab IgE Asthma ustekinumabIL12/IL23 Plaque psoriasis brodalumab IL17a Psoriasis, psoriaticarthritis, ankylosing spondylitis secukinumab IL17a Psoriasis, psoriaticarthritis, ankylosing spondylitis ixekizumab IL17a Psoriasis, psoriaticarthritis, ankylosing spondylitis canakinumab IL1β Cryopyrin-associatedperiodic syndrome, tumor necrosis factor receptor associated periodicsyndrome, hyperimmunoglobulin D syndrome, mevalonate kinase deficiency,familial Mediterranean fever, rheumatoid arthritis dupilumab IL4RαAsthma, dermatitis mepolizumab IL5 Asthma reslizumab IL5 Asthmatocilizumab IL6R Rheumatoid arthritis vedolizumab Integrin-α4β7Ulcerative colitis, Crohn's disease denosumab RANKL Osteoporosiscertolizumab TNFa Chron's disease, rheumatoid arthritis golimumab TNFaRheumatoid arthritis, psoriatic arthritis, ankylosing spondylitisadalimumab TNFα Rheumatoid arthritis, juvenile idiopathic arthritis,psoriatic arthritis ankylosing spondylitis, Crohn's disease, plaquepsoriasis infliximab TNFα Crohn's disease, ulcerative colitis,rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis,plaque psoriasis ranibizumab VEGF-A Neovascular age-related maculardegeneration, macular edema natalizumab VLA-4 Multiple sclerosis,relapsing rultiple sclerosis, Crohn's disease

The foregoing antibodies of Table 4 useful as supplementary agents inthe practice of the methods of the present disclosure may beadministered alone or in the form of any antibody drug conjugate (ADC)comprising the antibody, linker, and one or more drugs (e.g. 1, 2, 3, 4,5, 6, 7, or 8 drugs) or in modified form (e.g. PEGylated).

Formulations

The present disclosure further provides pharmaceutically acceptableformulations of the IFNgR2 binding molecules of the present disclosure.The preferred formulation depends on the intended mode of administrationand therapeutic application. Pharmaceutical dosage forms of the IFNgR2binding molecules described herein comprise physiologically acceptablecarriers that are inherently non-toxic and non-therapeutic. Examples ofsuch carriers include ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and PEG. Carriers for topical or gel-based forms ofpolypeptides include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG,polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

The pharmaceutical compositions may also comprisepharmaceutically-acceptable, non-toxic carriers, excipients,stabilizers, or diluents, which are defined as vehicles commonly used toformulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Acceptable carriers, excipients,or stabilizers are non-toxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyidimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Formulations to be used for in vivo administration are typicallysterile. Sterilization of the compositions of the present disclosure mayreadily accomplished by filtration through sterile filtration membranes.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). Theagents of this disclosure can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Vector Delivery of Polypeptide IFNgR2 Binding Molecules

In those embodiments where the IFNgR2 binding molecule is a polypeptide,such IFNgR2 binding molecules may also be delivered to a subject throughthe administration of a recombinant vectors comprising a nucleic acidsequence encoding the peptidyl IFNgR2 binding molecule operably linkedto an expression control sequence in the cells of the tissues of thesubject.

Expression vectors may be viral vectors or non-viral vectors. The term“nonviral vector” refers to an autonomously replicating,extrachromosomal circular DNA molecule, distinct from the normal genomeand nonessential for cell survival under nonselective conditions capableof effecting the expression of an coding sequence in the target cell.Plasmids are examples of non-viral vectors. In order to facilitatetransfection of the target cells, the target cell may be exposeddirectly with the non-viral vector may under conditions that facilitateuptake of the non-viral vector. Examples of conditions which facilitateuptake of foreign nucleic acid by mammalian cells are well known in theart and include but are not limited to chemical means (such asLipofectamine®, Thermo-Fisher Scientific), high salt, magnetic fields(electroporation)

In one embodiment, a non-viral vector may be provided in a non-viraldelivery system. Non-viral delivery systems are typically complexes tofacilitate transduction of the target cell with a nucleic acid cargowherein the nucleic acid is complexed with agents such as cationiclipids (DOTAP, DOTMA), surfactants, biologicals (gelatin, chitosan),metals (gold, magnetic iron) and synthetic polymers (PLG, PEI, PAMAM).Numerous embodiments of non-viral delivery systems are well known in theart including lipidic vector systems (Lee et al. (1997) Crit Rev TherDrug Carrier Syst. 14:173-206); polymer coated liposomes (Marin et al.,U.S. Pat. No. 5,213,804, issued May 25, 1993; Woodle, et al., U.S. Pat.No. 5,013,556, issued May 7, 1991); cationic liposomes (Epand et al.,U.S. Pat. No. 5,283,185, issued Feb. 1, 1994; Jessee, J. A., U.S. Pat.No. 5,578,475, issuedNov. 26, 1996; Rose et al, U.S. Pat. No. 5,279,833,issued Jan. 18, 1994; Gebeyehu et al., U.S. Pat. No. 5,334,761, issuedAug. 2, 1994).

In another embodiment, the expression vector may be a viral vector. Asused herein, the term viral vector is used in its conventional sense torefer to any of the obligate intracellular parasites having noprotein-synthesizing or energy-generating mechanism and generally refersto any of the enveloped or non-enveloped animal viruses commonlyemployed to deliver exogenous transgenes to mammalian cells. A viralvector may be replication competent (e.g., substantially wild-type),conditionally replicating (recombinantly engineered to replicate undercertain conditions) or replication deficient (substantially incapable ofreplication in the absence of a cell line capable of complementing thedeleted functions of the virus). The viral vector can possess certainmodifications to make it “specifically replicating,” i.e. that itreplicates preferentially in certain cell types or phenotypic cellstates, e.g., cancerous. Viral vector systems useful in the practice ofthe instant IFNgR2 binding molecule include, for example, naturallyoccurring or recombinant viral vector systems. Examples of virusesuseful in the practice of the present IFNgR2 binding molecule includerecombinantly modified enveloped or non-enveloped DNA and RNA viruses.For example, viral vectors can be derived from the genome of human orbovine adenoviruses, vaccinia virus, lentivirus, herpes virus,adeno-associated virus, human immunodeficiency virus, sindbis virus, andretroviruses (including but not limited to Rous sarcoma virus), andhepatitis B virus. Typically, genes of interest are inserted into suchvectors to allow packaging of the gene construct, typically withaccompanying viral genomic sequences, followed by infection of asensitive host cell resulting in expression of the gene of interest(e.g., a targeting antigen).

The expression vector may encode one or more polypeptides in addition tothe targeting antigen. When expressing multiple polypeptides as in thepractice of the present IFNgR2 binding molecule, each polypeptide may beoperably linked to an expression control sequence (monocistronic) ormultiple polypeptides may be encoded by a polycistronic construct wheremultiple polypeptides are expressed under the control of a singleexpression control sequence. In one embodiment, the expression vectorencoding the targeting antigen may optionally further encode one or moreimmunological modulators. Examples of immunological modulators useful inthe practice of the present IFNgR2 binding molecule include but are notlimited to cytokines. Examples of such cytokines are interleukinsincluding but not limited to one more or of IL-1, IL-2, IL-3, IL-4,IL-12, TNF-alpha, interferon alpha, interferon alpha-2b,interferon-beta, interferon-gamma, GM-CSF, MIP1-alpha, MIP1-beta,MIP3-alpha, TGF-beta and other suitable cytokines capable of modulatingimmune response. The expressed cytokines can be directed forintracellular expression or expressed with a signal sequence forextracellular presentation or secretion.

The expression vector may optionally provide an additional expressioncassette comprising a nucleic acid sequence encoding a “rescue” gene. A“rescue gene” is a nucleic acid sequence, the expression of whichrenders the cell susceptible to killing by external factors or causes atoxic condition in the cell such that the cell is killed. Providing arescue gene enables selective cell killing of transduced cells. Thus,the rescue gene provides an additional safety precaution when saidconstructs are incorporated into the cells of a mammalian subject toprevent undesirable spreading of transduced cells or the effects ofreplication competent vector systems. In one embodiment, the rescue geneis the thymidine kinase (TK) gene (see e.g., Woo, et al. U.S. Pat. No.5,631,236 issued May 20, 1997 and Freeman, et al. U.S. Pat. No.5,601,818 issued Feb. 11, 1997) in which the cells expressing the TKgene product are susceptible to selective killing by the administrationof gancyclovir.

Dosage

The present disclosure further provides the administration oftherapeutically or prophylactically effective dose of IFNgR2 bindingmolecule or a recombinant vector or cell comprising a nucleic acidsequence encoding a polypeptide IFNgR2 binding molecule to a subjectsuffering from or at risk of developing, respectively, a disease,disorder or condition. The dosage of the pharmaceutical compositioncomprising the IFNgR2 binding molecules, vector or cell depends onfactors including the route of administration, the disease to betreated, and physical characteristics, e.g., age, weight, generalhealth, of the subject. Typically, the amount of a IFNgR2 bindingmolecule contained within a single dose may be an amount thateffectively prevents, delays, or treats the disease without inducingsignificant toxicity. A pharmaceutical composition of the disclosure mayinclude a dosage of a IFNgR2 binding molecule described herein rangingfrom 0.01 to 500 mg/kg (e.g., from 0.01 to 450 mg, from 0.01 to 400 mg,from 0.01 to 350 mg, from 0.01 to 300 mg, from 0.01 to 250 mg, from 0.01to 200 mg, from 0.01 to 150 mg, from 0.01 to 100 mg, from 0.01 to 50 mg,from 0.01 to 10 mg, from 0.01 to 1 mg, from 0.1 to 500 mg/kg, from 1 to500 mg/kg, from 5 to 500 mg/kg, from 10 to 500 mg/kg, from 50 to 500mg/kg, from 100 to 500 mg/kg, from 150 to 500 mg/kg, from 200 to 500mg/kg, from 250 to 500 mg/kg, from 300 to 500 mg/kg, from 350 to 500mg/kg, from 400 to 500 mg/kg, or from 450 to 500 mg/kg) and, in a morespecific embodiment, about 1 to about 100 mg/kg (e.g., about 1 to about90 mg/kg, about 1 to about 80 mg/kg, about 1 to about 70 mg/kg, about 1to about 60 mg/kg, about 1 to about 50 mg/kg, about 1 to about 40 mg/kg,about 1 to about 30 mg/kg, about 1 to about 20 mg/kg, about 1 to about10 mg/kg, about 10 to about 100 mg/kg, about 20 to about 100 mg/kg,about 30 to about 100 mg/kg, about 40 to about 100 mg/kg, about 50 toabout 100 mg/kg, about 60 to about 100 mg/kg, about 70 to about 100mg/kg, about 80 to about 100 mg/kg, or about 90 to about 100 mg/kg). Insome embodiments, a pharmaceutical composition of the disclosure mayinclude a dosage of a binding protein described herein ranging from 0.01to 20 mg/kg (e.g., from 0.01 to 15 mg/kg, from 0.01 to 10 mg/kg, from0.01 to 8 mg/kg, from 0.01 to 6 mg/kg, from 0.01 to 4 mg/kg, from 0.01to 2 mg/kg, from 0.01 to 1 mg/kg, from 0.01 to 0.1 mg/kg, from 0.01 to0.05 mg/kg, from 0.05 to 20 mg/kg, from 0.1 to 20 mg/kg, from 1 to 20mg/kg, from 2 to 20 mg/kg, from 4 to 20 mg/kg, from 6 to 20 mg/kg, from8 to 20 mg/kg, from 10 to 20 mg/kg, from 15 to 20 mg/kg). The dosage maybe adapted by the physician in accordance with conventional factors suchas the extent of the disease and different parameters of the subject.

A pharmaceutical composition containing a IFNgR2 binding moleculedescribed herein can be administered to a subject in need thereof, forexample, one or more times (e.g., 1-10 times or more) daily, weekly,monthly, biannually, annually, or as medically necessary. Dosages may beprovided in either a single or multiple dosage regimens. The timingbetween administrations may decrease as the medical condition improvesor increase as the health of the patient declines. A course of therapymay be a single dose or in multiple doses over a period of time. In someembodiments, a single dose is used. In some embodiments, two or moresplit doses administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 21, 28, 30, 60, 90, 120 or 180 days are used. Each doseadministered in such split dosing protocols may be the same in eachadministration or may be different. Multi-day dosing protocols over timeperiods may be provided by the skilled artisan (e.g., physician)monitoring the administration, taking into account the response of thesubject to the treatment including adverse effects of the treatment andtheir modulation as discussed above.

For prophylactic applications, pharmaceutical compositions ormedicaments are administered to a patient susceptible to, or otherwiseat risk of disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease.

In some embodiments the condition to be treated is a chronic condition(e.g., a chronic infection, i.e., an infection that is not cleared bythe host immune system within a period of up to 1 week, 2 weeks, etc.).In some cases, chronic condition involve integration of pathogen geneticelements into the host genome, e.g., retroviruses, lentiviruses,Hepatitis B virus, etc. In other cases, chronic infections, for examplecertain intracellular bacteria or protozoan pathogens, result from apathogen cell residing within a host cell. Additionally, in someembodiments, the infection is in a latent stage, as with herpes virusesor human papilloma viruses. In such instances, the course of therapy mayinvolve the administration of the IFNgR2 binding molecule over anextended period of time including continued administration in thesubstantial absence of the symptoms of the chronic condition to preventrecurrence of the chronic conditions or symptoms thereof.

In prophylactic applications, a relatively low dosage may beadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In other therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patent can be administered a prophylactic regime.

Formulations

The present disclosure further provides pharmaceutically acceptableformulations of the IFNgR2 binding molecules of the present disclosure.The preferred formulation depends on the intended mode of administrationand therapeutic application. Pharmaceutical dosage forms of the IFNgR2binding molecules described herein comprise physiologically acceptablecarriers that are inherently non-toxic and non-therapeutic. Examples ofsuch carriers include ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and PEG. Carriers for topical or gel-based forms ofpolypeptides include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG,polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

The pharmaceutical compositions may also comprisepharmaceutically-acceptable, non-toxic carriers, excipients,stabilizers, or diluents, which are defined as vehicles commonly used toformulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Acceptable carriers, excipients,or stabilizers are non-toxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyidimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Formulations to be used for in vivo administration are typicallysterile. Sterilization of the compositions of the present disclosure mayreadily accomplished by filtration through sterile filtration membranes.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). Theagents of this disclosure can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Vector Delivery of Polypeptide IFNgR2 Binding Molecules

In those embodiments where the IFNgR2 binding molecule is a polypeptide,such IFNgR2 binding molecules may also be delivered to a subject throughthe administration of a recombinant vectors comprising a nucleic acidsequence encoding the peptidyl IFNgR2 binding molecule operably linkedto an expression control sequence in the cells of the tissues of thesubject.

Expression vectors may be viral vectors or non-viral vectors. The term“nonviral vector” refers to an autonomously replicating,extrachromosomal circular DNA molecule, distinct from the normal genomeand nonessential for cell survival under nonselective conditions capableof effecting the expression of an coding sequence in the target cell.Plasmids are examples of non-viral vectors. In order to facilitatetransfection of the target cells, the target cell may be exposeddirectly with the non-viral vector may under conditions that facilitateuptake of the non-viral vector. Examples of conditions which facilitateuptake of foreign nucleic acid by mammalian cells are well known in theart and include but are not limited to chemical means (such asLipofectamine®, Thermo-Fisher Scientific), high salt, magnetic fields(electroporation)

In one embodiment, a non-viral vector may be provided in a non-viraldelivery system. Non-viral delivery systems are typically complexes tofacilitate transduction of the target cell with a nucleic acid cargowherein the nucleic acid is complexed with agents such as cationiclipids (DOTAP, DOTMA), surfactants, biologicals (gelatin, chitosan),metals (gold, magnetic iron) and synthetic polymers (PLG, PEI, PAMAM).Numerous embodiments of non-viral delivery systems are well known in theart including lipidic vector systems (Lee et al. (1997) Crit Rev TherDrug Carrier Syst. 14:173-206); polymer coated liposomes (Marin et al.,U.S. Pat. No. 5,213,804, issued May 25, 1993; Woodle, et al., U.S. Pat.No. 5,013,556, issued May 7, 1991); cationic liposomes (Epand et al.,U.S. Pat. No. 5,283,185, issued Feb. 1, 1994; Jessee, J. A., U.S. Pat.No. 5,578,475, issuedNov. 26, 1996; Rose et al, U.S. Pat. No. 5,279,833,issued Jan. 18, 1994; Gebeyehu et al., U.S. Pat. No. 5,334,761, issuedAug. 2, 1994).

In another embodiment, the expression vector may be a viral vector. Asused herein, the term viral vector is used in its conventional sense torefer to any of the obligate intracellular parasites having noprotein-synthesizing or energy-generating mechanism and generally refersto any of the enveloped or non-enveloped animal viruses commonlyemployed to deliver exogenous transgenes to mammalian cells. A viralvector may be replication competent (e.g., substantially wild-type),conditionally replicating (recombinantly engineered to replicate undercertain conditions) or replication deficient (substantially incapable ofreplication in the absence of a cell line capable of complementing thedeleted functions of the virus). The viral vector can possess certainmodifications to make it “specifically replicating,” i.e. that itreplicates preferentially in certain cell types or phenotypic cellstates, e.g., cancerous. Viral vector systems useful in the practice ofthe instant IFNgR2 binding molecule include, for example, naturallyoccurring or recombinant viral vector systems. Examples of virusesuseful in the practice of the present IFNgR2 binding molecule includerecombinantly modified enveloped or non-enveloped DNA and RNA viruses.For example, viral vectors can be derived from the genome of human orbovine adenoviruses, vaccinia virus, lentivirus, herpes virus,adeno-associated virus, human immunodeficiency virus, sindbis virus, andretroviruses (including but not limited to Rous sarcoma virus), andhepatitis B virus. Typically, genes of interest are inserted into suchvectors to allow packaging of the gene construct, typically withaccompanying viral genomic sequences, followed by infection of asensitive host cell resulting in expression of the gene of interest(e.g., a targeting antigen).

The expression vector may encode one or more polypeptides in addition tothe targeting antigen. When expressing multiple polypeptides as in thepractice of the present IFNgR2 binding molecule, each polypeptide may beoperably linked to an expression control sequence (monocistronic) ormultiple polypeptides may be encoded by a polycistronic construct wheremultiple polypeptides are expressed under the control of a singleexpression control sequence. In one embodiment, the expression vectorencoding the targeting antigen may optionally further encode one or moreimmunological modulators. Examples of immunological modulators useful inthe practice of the present IFNgR2 binding molecule include but are notlimited to cytokines. Examples of such cytokines are interleukinsincluding but not limited to one more or of IL-1, IL-2, IL-3, IL-4,IL-12, TNF-alpha, interferon alpha, interferon alpha-2b,interferon-beta, interferon-gamma, GM-CSF, MIP1-alpha, MIP1-beta,MIP3-alpha, TGF-beta and other suitable cytokines capable of modulatingimmune response. The expressed cytokines can be directed forintracellular expression or expressed with a signal sequence forextracellular presentation or secretion.

The expression vector may optionally provide an additional expressioncassette comprising a nucleic acid sequence encoding a “rescue” gene. A“rescue gene” is a nucleic acid sequence, the expression of whichrenders the cell susceptible to killing by external factors or causes atoxic condition in the cell such that the cell is killed. Providing arescue gene enables selective cell killing of transduced cells. Thus,the rescue gene provides an additional safety precaution when saidconstructs are incorporated into the cells of a mammalian subject toprevent undesirable spreading of transduced cells or the effects ofreplication competent vector systems. In one embodiment, the rescue geneis the thymidine kinase (TK) gene (see e.g., Woo, et al. U.S. Pat. No.5,631,236 issued May 20, 1997 and Freeman, et al. U.S. Pat. No.5,601,818 issued Feb. 11, 1997) in which the cells expressing the TKgene product are susceptible to selective killing by the administrationof gancyclovir.

Dosage

The present disclosure further provides the administration oftherapeutically or prophylactically effective dose of IFNgR2 bindingmolecule or a recombinant vector or cell comprising a nucleic acidsequence encoding a polypeptide IFNgR2 binding molecule to a subjectsuffering from or at risk of developing, respectively, a disease,disorder or condition. The dosage of the pharmaceutical compositioncomprising the IFNgR2 binding molecules, vector or cell depends onfactors including the route of administration, the disease to betreated, and physical characteristics, e.g., age, weight, generalhealth, of the subject. Typically, the amount of a IFNgR2 bindingmolecule contained within a single dose may be an amount thateffectively prevents, delays, or treats the disease without inducingsignificant toxicity. A pharmaceutical composition of the disclosure mayinclude a dosage of a IFNgR2 binding molecule described herein rangingfrom 0.01 to 500 mg/kg (e.g., from 0.01 to 450 mg, from 0.01 to 400 mg,from 0.01 to 350 mg, from 0.01 to 300 mg, from 0.01 to 250 mg, from 0.01to 200 mg, from 0.01 to 150 mg, from 0.01 to 100 mg, from 0.01 to 50 mg,from 0.01 to 10 mg, from 0.01 to 1 mg, from 0.1 to 500 mg/kg, from 1 to500 mg/kg, from 5 to 500 mg/kg, from 10 to 500 mg/kg, from 50 to 500mg/kg, from 100 to 500 mg/kg, from 150 to 500 mg/kg, from 200 to 500mg/kg, from 250 to 500 mg/kg, from 300 to 500 mg/kg, from 350 to 500mg/kg, from 400 to 500 mg/kg, or from 450 to 500 mg/kg) and, in a morespecific embodiment, about 1 to about 100 mg/kg (e.g., about 1 to about90 mg/kg, about 1 to about 80 mg/kg, about 1 to about 70 mg/kg, about 1to about 60 mg/kg, about 1 to about 50 mg/kg, about 1 to about 40 mg/kg,about 1 to about 30 mg/kg, about 1 to about 20 mg/kg, about 1 to about10 mg/kg, about 10 to about 100 mg/kg, about 20 to about 100 mg/kg,about 30 to about 100 mg/kg, about 40 to about 100 mg/kg, about 50 toabout 100 mg/kg, about 60 to about 100 mg/kg, about 70 to about 100mg/kg, about 80 to about 100 mg/kg, or about 90 to about 100 mg/kg). Insome embodiments, a pharmaceutical composition of the disclosure mayinclude a dosage of a binding protein described herein ranging from 0.01to 20 mg/kg (e.g., from 0.01 to 15 mg/kg, from 0.01 to 10 mg/kg, from0.01 to 8 mg/kg, from 0.01 to 6 mg/kg, from 0.01 to 4 mg/kg, from 0.01to 2 mg/kg, from 0.01 to 1 mg/kg, from 0.01 to 0.1 mg/kg, from 0.01 to0.05 mg/kg, from 0.05 to 20 mg/kg, from 0.1 to 20 mg/kg, from 1 to 20mg/kg, from 2 to 20 mg/kg, from 4 to 20 mg/kg, from 6 to 20 mg/kg, from8 to 20 mg/kg, from 10 to 20 mg/kg, from 15 to 20 mg/kg). The dosage maybe adapted by the physician in accordance with conventional factors suchas the extent of the disease and different parameters of the subject.

A pharmaceutical composition containing a IFNgR2 binding moleculedescribed herein can be administered to a subject in need thereof, forexample, one or more times (e.g., 1-10 times or more) daily, weekly,monthly, biannually, annually, or as medically necessary. Dosages may beprovided in either a single or multiple dosage regimens. The timingbetween administrations may decrease as the medical condition improvesor increase as the health of the patient declines. A course of therapymay be a single dose or in multiple doses over a period of time. In someembodiments, a single dose is used. In some embodiments, two or moresplit doses administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 21, 28, 30, 60, 90, 120 or 180 days are used. Each doseadministered in such split dosing protocols may be the same in eachadministration or may be different. Multi-day dosing protocols over timeperiods may be provided by the skilled artisan (e.g., physician)monitoring the administration, taking into account the response of thesubject to the treatment including adverse effects of the treatment andtheir modulation as discussed above.

For prophylactic applications, pharmaceutical compositions ormedicaments are administered to a patient susceptible to, or otherwiseat risk of disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease.

In some embodiments the condition to be treated is a chronic condition(e.g., a chronic infection, i.e., an infection that is not cleared bythe host immune system within a period of up to 1 week, 2 weeks, etc.).In some cases, chronic condition involve integration of pathogen geneticelements into the host genome, e.g., retroviruses, lentiviruses,Hepatitis B virus, etc. In other cases, chronic infections, for examplecertain intracellular bacteria or protozoan pathogens, result from apathogen cell residing within a host cell. Additionally, in someembodiments, the infection is in a latent stage, as with herpes virusesor human papilloma viruses. In such instances, the course of therapy mayinvolve the administration of the IFNgR2 binding molecule over anextended period of time including continued administration in thesubstantial absence of the symptoms of the chronic condition to preventrecurrence of the chronic conditions or symptoms thereof.

In prophylactic applications, a relatively low dosage may beadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In other therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patent can be administered a prophylactic regime.

Routes of Administration

Administration of a IFNgR2 binding molecules described herein may beachieved through any of a variety of art recognized methods includingbut not limited to the topical, intravascular injection (includingintravenous or intraarterial infusion), intradermal injection,subcutaneous injection, intramuscular injection, intraperitonealinjection, intracranial injection, intratumoral injection, intranodalinjection, transdermal, transmucosal, iontophoretic delivery,intralymphatic injection (Senti and Kundig (2009) Current Opinions inAllergy and Clinical Immunology 9(6):537-543), intragastric infusion,intraprostatic injection, intravesical infusion (e.g., bladder),respiratory inhalers including nebulizers, intraocular injection,intraabdominal injection, intralesional injection, intraovarianinjection, intracerebral infusion or injection, intracerebroventricularinjection (ICVI), and the like. Administration to the subject may beachieved by intravenous, as a bolus or by continuous infusion over aperiod of time. Examples of parenteral routes of administration include,for example, intravenous, intradermal, subcutaneous, transdermal(topical), transmucosal, and rectal administration. The IFNgR2 bindingmolecule can be administered once, continuously, such as by continuouspump, or at periodic (e.g., daily, bi-weekly, monthly) intervals over aperiod of time can occur over the period of one week, two weeks, onemonth, two months, three months or more. Desired time intervals ofmultiple doses of the IFNgR2 binding molecule may be determined by oneof skill in the art.

As described hereinabove, the compositions of the present disclosure maybe used in combination with one or more additional therapeuticallyeffective agents. As used herein, the term “in combination with” whenused in reference to the administration of multiple agents to a subjectrefers to the administration of a first agent at least one additional(i.e. second, third, fourth, fifth, etc.) supplementary agent to asubject. For purposes of the present disclosure, one agent (e.g., aIFNgR2 binding molecule) is considered to be administered in combinationwith a supplementary agent if the biological effect resulting from theadministration of the first agent persists in the subject at the time ofadministration of the supplementary agent such that the therapeuticeffects of the first agent and second agent overlap. The administrationof the first agent may provide a therapeutic effect over an extendedtime and the administration of the supplementary agent provides itstherapeutic effect while the therapeutic effect of the first agentremains ongoing such that the supplementary agent is considered to beadministered in combination with the first agent, even though the firstagent may have been administered at a point in time significantlydistant (e.g., days or weeks) from the time of administration of thesupplementary agent. In one embodiment, one agent is considered to beadministered in combination with a supplementary agent if the first andsecond agents are administered simultaneously (within 30 minutes of eachother), contemporaneously or sequentially. In some embodiments, a firstagent is deemed to be administered “contemporaneously” with asupplementary agent if first and supplementary agents are administeredwithin about 24 hours of each another, preferably within about 12 hoursof each other, preferably within about 6 hours of each other, preferablywithin about 2 hours of each other, or preferably within about 30minutes of each other. The term “in combination with” shall alsounderstood to apply to the situation where a first agent and asupplementary agent are co-formulated in single pharmaceuticallyacceptable formulation and the co-formulation is administered to asubject. In certain embodiments, first agent and the supplementaryagent(s) are administered or applied sequentially, e.g., where one agentis administered prior to one or more other agents. In other embodiments,the first agent and the supplementary agent(s) are administeredsimultaneously, for example where two or more agents are administered ator about the same time; the two or more agents may be present in two ormore separate formulations or combined into a single formulation (i.e.,a co-formulation). Regardless of whether the agents are administeredsequentially or simultaneously, they are considered to be administeredin combination for purposes of the present disclosure.

Kits

The present disclosure also contemplates kits comprising pharmaceuticalcompositions of IFNgR2 binding molecules. In some embodiments, the kitfurther comprises supplementary pharmaceutical compositions comprisingsupplementary agents as discussed above for use in combination therapywith IFNgR2 binding molecules. The kits are generally in the form of aphysical structure housing various components, as described below, andcan be utilized, for example, in practicing the methods described above.A kit may comprise a IFNgR2 binding molecule in the form of apharmaceutical composition suitable for administration to a subject thatis ready for use or in a form or requiring preparation for example,thawing, reconstitution or dilution prior to administration. When theIFNgR2 binding molecule is in a form that requires reconstitution by auser, the kit may also comprise a sterile container providing areconstitution medium comprising buffers, pharmaceutically acceptableexcipients, and the like. A kit of the present disclosure can bedesigned for conditions necessary to properly maintain the componentshoused therein (e.g., refrigeration or freezing). A kit may furthercontain a label or packaging insert including identifying informationfor the components therein and instructions for their use. Eachcomponent of the kit can be enclosed within an individual container, andall of the various containers can be within a single package. Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert can be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampule, syringe or vial). Labels or inserts may be provided ina physical form or a computer readable medium. In some embodiments, theactual instructions are not present in the kit, but rather the kitprovides a means for obtaining the instructions from a remote source,e.g., via an internet site, including by secure access by providing apassword (or scannable code such as a barcode or QR code on thecontainer of the IFNgR2 binding molecule or kit comprising) incompliance with governmental regulations (e.g., HIPAA) are provided.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present IFNgR2 binding molecule, and are not intendedto limit the scope of what the inventors regard as their IFNgR2 bindingmolecule nor are they intended to represent that the experiments belowwere performed and are all of the experiments that can be performed. Itis to be understood that exemplary descriptions written in the presenttense were not necessarily performed, but rather that the descriptionscan be performed to generate the data and the like described therein.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.), but some experimental errors anddeviations should be accounted for. Variations of the particularlydescribed procedures employed may become apparent to individuals orskill in the art and it is expected that those skilled artisans mayemploy such variations as appropriate. Accordingly, it is intended thatthe IFNgR2 binding molecule be practiced otherwise than as specificallydescribed herein, and that the invention includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: bp=base pair(s); kb=kilobase(s);pl=picoliter(s); s or sec=second(s); min=minute(s); h or hr=hour(s);aa=amino acid(s); kb=kilobase(s); nt=nucleotide(s); pg=picogram;ng=nanogram; pg=microgram; mg=milligram; g=gram; kg=kilogram; dl ordL=deciliter; pl or pL=microliter; ml or mL=milliliter; 1 or L=liter;pM=micromolar; mM=millimolar; M=molar; kDa=kilodalton;i.m.=intramuscular(ly); i.p.=intraperitoneal(ly); SC orSQ=subcutaneous(ly); QD=daily; BID=twice daily; QW=weekly; QM=monthly;HPLC=high performance liquid chromatography; BW=body weight; U=unit;ns=not statistically significant; PBS=phosphate-buffered saline;PCR=polymerase chain reaction; NHS=N-hydroxysuccinimide; HSA=human serumalbumin; MSA=mouse serum albumin; DMEM=Dulbeco's Modification of Eagle'sMedium; GC=genome copy; EDTA=ethylenediaminetetraacetic acid;PBMCs=primary peripheral blood mononuclear cells; FBS=fetal bovineserum; FCS=fetal calf serum;HEPES=4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid;LPS=lipopolysaccharide; ATCC=American Type Culture Collection

Example 1. Immunization Protocol

The VHH was obtained by immunization of a llama with the extracellulardomains of the hIFNgR2 as the antigen. A synthetic DNA sequence encodingthe antigen was inserted into the pExSyn2.0 vector and transfected intothe HEK293F mammalian cell host cell for expression. The antigen isexpressed as a his tagged fusion protein which is purified usingimmobilized metal affinity chromatography. The antigen was diluted with1 xPBS (antigen total about 1 mg). The quality was estimated by SDS-PAGEto ensure the purity was sufficient (>90%) for immunization. The llamawas acclimated at the facility for atleast 7 days. At the start of theimmunization protocol, 100 mL of blood was drawn to be used as apre-bleed followed by boost with 150 to 500 ug of protein. Additionalboosts were performed on days 14, 28, 42, and 56. 350 ml of blood wascollected on day 63 for construction of the yeast sdAb library.

Example 2. Yeast Library Construction

350 ml of blood sample was collected from the llama seven days followingthe last injection in the immunization protocol. RNA was extracted fromblood and transcribed to cDNA. The approximately 900 bp reversetranscribed sequences encoding the VH-CH1-hinge-CH2-CH3 constructs wereisolated from the approximately desired 700 bp fragments encoding theVHH-hinge-CH2-CH3 species. The purified approximately 700 bp fragmentswere amplified by nested PCR. The amplified fragments were inserted intoa NheI/BamHI digested pGAL 414 yeast display vector such that thesequence encoding the VHH was fused to the C-term of Aga2 with an N-termHA and C-term Myc tag. Yeast cells were transformed with digested vectorand amplified insert. Transformants were enriched in SD-SCAA medialacking trptophan and uracil and stored at 4C till further use.

Example 3: Isolation of Antigen Specific VHHs

sdAb were expressed on the surface of yeast (S. cervisiae ATCC strainEBY100) using standard protocols. Yeast were grown overnight at 30° C.in synthetic selective media SD-SCAA, then induced again overnight at20° C. in SG-SCAA. SdAb expressing yeast were incubated with Alexa647conjugated IFNGR1 from 30 minutes to 2 hours. Antigen binding yeast weresorted using a Sony sorter (SH800). After 3 rounds of selection, yeastcells were plated onto SD-SCAA plates lacking tryptophan and uracil. 192colonies were picked from each campaign and grown overnight in 96 wellplates at 30° C. in synthetic selective media SD-SCAA, then inducedagain overnight at 20° C. in SG-SCAA.

Example 4: Identification of Antibodies Exhibiting Specific Binding toIFNgR2

Upon completion of the biopanning of Example 3, three 96-well plates ofindividual phage clones were isolated in order to perform periplasmicextract ELISA (PE-ELISA) on IFNgR2 coated plates to identify positiveVHH binders that selectively bound IFNgR2. A 96-well plate was coatedwith IFNgR2 and PBS under the same conditions. Next, wells were blockedat 37° C. for 1 h. Then, 100 μl of extracted antibodies was added toeach well and incubated for 1 h. Subsequently, 100 μl of anti-tagpolyclonal antibody conjugated to HRP was added to each well andincubated at 37° C. for 1 h. Plates were developed with TMB substrate.The reaction was stopped by the addition of H2SO4. Absorbance at 450 nmwas read on a microtiter plate reader. Antibodies with absorbance of theantigen-coated well at least threefold greater than PBS-coated controlwere considered to provide specific binding to IFNgR2. Positive cloneswere sequenced, and sequences analyzed to identify unique clonotypes

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, sequence accessionnumbers, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

1. A IFNgR2 binding molecule that specifically binds to theextracellular domain of IFNgR2.
 2. The IFNgR2 binding molecule of claim1, wherein the IFNgR2 binding molecule comprises a single domainantibody (sdAb).
 3. The IFNgR2 binding molecule of claim 2, wherein thesdAb comprises a complementary determining region 1 (CDR1), a CDR2, anda CDR3 as shown in a row of the table below: CDR1 CDR2 CDR3 RTFTSYAMNAISWSSGNTYVADSVKG TTIATMSDEYTY (SEQ ID NO: 34) (SEQ ID NO: 35)(SEQ ID NO: 36) RTFSNYRMG AISRGGGTTLYADSVKG GDFSTTWDEYNY (SEQ ID NO: 37)(SEQ ID NO: 38) (SEQ ID NO: 39) RTFSSYAMG AISRGGGSTDYADSVKGRAYSGRYYQFLEYDY (SEQ ID NO: 40) (SEQ ID NO: 41) (SEQ ID NO: 42)RTFSSYAVA ALSRGGGSAYYTDSVKG RNYDGTYYQENQYNY (SEQ ID NO: 43)(SEQ ID NO : 44) (SEQ ID NO: 45) RTFSTYAMG AISVNGGSTYYADSVTGRRPYPGSDFLTWASYDY (SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48)RTFSYTTIG VISAGGGSRDYADALKG RRNTDTYTTTGDYDY (SEQ ID NO: 49)(SEQ ID NO: 50) (SEQ ID NO: 51) GTISSLAMG AISWSGRSTYYVDSVKG GEDGHSEYDY(SEQ ID NO: 52) (SEQ ID NO: 53) (SEQ ID NO: 54) RSFANYAMGAISRGGGSTWYADSVKG RSYSGSYTYSFGEYDY (SEQ ID NO: 55) (SEQ ID NO: 56)(SEQ ID NO: 57) RAFSTYALG AISRGGGSTDYADSVKG RSYSSSYYYSQYEYDY(SEQ ID NO: 58) (SEQ ID NO: 59) (SEQ ID NO: 60) RTFSSVAMAAISSGGGSTDYADSVKG RDYSSRRYYQSRYEYDL (SEQ ID NO: 61) (SEQ ID NO : 62)(SEQ ID NO: 63) RTFRSYSMG AISWYSGTTYYADPVKG NEIATMESSNDY (SEQ ID NO: 64)(SEQ ID NO: 65) (SEQ ID NO: 66) RPRTTYAMG AISKAGGSTYVADSAKGRAGFAAQIFEYDY (SEQ ID NO: 67) (SEQ ID NO: 68) (SEQ ID NO: 69) RTFGTYAMGSIDRDGSMSYYADSVKG SRRAVISLQTVDY (SEQ ID NO: 70) (SEQ ID NO: 71)(SEQ ID NO: 72) RTFSNYAMG AISWYSGNTYYADSVKG NQIATMISVGDY (SEQ ID NO: 73)(SEQ ID NO: 74) (SEQ ID NO: 75) SIFRLNLMG HVGTTGNTAYADSVKG DRWGQFS(SEQ ID NO: 76) (SEQ ID NO: 77) (SEQ ID NO: 78) RTFTGYAMGVITWSGATTYYSASVKG RIRDGVSPENPNEYGY (SEQ ID NO: 79) (SEQ ID NO: 80)(SEQ ID NO: 81) RTVGYGMA AITWSGTSTYYPDSVKG GSRRRVGVDVGGYDY(SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) RTFTFSTHNMGGIMWTSRASYADSVKG AWYGNSGASYDY (SEQ ID NO: 85) (SEQ ID NO : 86)(SEQ ID NO: 87) RTFSGYNMG AIAWAGSRTYYTDSVKG HDET YYRLDRVDLYTH(SEQ ID NO: 88) (SEQ ID NO: 89) (SEQ ID NO: 90) PFTRYAMGAISWSSGNTYYVDSVKG NEVATMSGPDDY (SEQ ID NO: 91) (SEQ ID NO: 92)(SEQ ID NO: 93) GSFGRYTMG VISWSGTNTYYADSVKG RETYYSHWDERMEYDY(SEQ ID NO: 94) (SEQ ID NO : 95) (SEQ ID NO: 96) SIDSSYYVSAINWGDSRTAYADSVKG RIGLGGPVVAAPTRYPY (SEQ ID NO: 97) (SEQ ID NO: 98)(SEQ ID NO : 99) SILRFNVMS VITSGGSTNYADSVKG DESGQYY (SEQ ID NO: 100)(SEQ ID NO : 101) (SEQ ID NO: 102) LTTSSAALA TITSGGGSTYYADSVKGRFYSTTYYYREHEYSD (SEQ ID NO: 103) (SEQ ID NO: 104) (SEQ ID NO: 105)RTFGTSFGSLSMG AISRNIGRTYYADSVKD TNSYDDLRRSYAYDY (SEQ ID NO: 106)(SEQ ID NO : 107) (SEQ ID NO: 108) RTFSSLAMA AISRSGGSTDYADSVKGRDYSTLQYYNEYEYSD (SEQ ID NO: 109) (SEQ ID NO: 110) (SEQ ID NO : 111)PTFSTYAMA AITQSGRTTYYEDSVKG RDLWSDSPDDWRIYSF (SEQ ID NO: 112)(SEQ ID NO: 113) (SEQ ID NO : 114) RTLHNFAMA AISKGGGSADYADSVKGNDLASYSDSSYTSTSRYDY (SEQ ID NO: 115) (SEQ ID NO : 116) (SEQ ID NO : 117)RTFSSYAMA AISILGGSADYEDSVQG RRPAPSDSYWSSTSYAY (SEQ ID NO: 118)(SEQ ID NO : 119) (SEQ ID NO : 120) RTFSSLAMA ATTILGGSADYGDPVKGRRPAPSDNYWSPASYAY (SEQ ID NO: 121) (SEQ ID NO: 122) (SEQ ID NO : 123)RTFSSYAMA AITVSGASTYYADSVKG GGPGTIFPDYDY (SEQ ID NO: 124)(SEQ ID NO: 125) (SEQ ID NO : 126) RTFSGYNMG AINWIGGATYYADSVKGYSEKFYSGKDYYTRDYDY (SEQ ID NO: 127) (SEQ ID NO : 128) (SEQ ID NO: 129)


4. The IFNgR2 binding molecule of claim 2, wherein the sdAb has at least80%, alternatively at least 85%, alternatively at least 90%,alternatively at least 95% alternatively at least 98%, alternatively atleast 99% identity, or 100% identity to a polypeptide sequence of anyone of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33.5. The IFNgR2 binding molecule of claim 3, wherein the sdAb is humanizedor otherwise comprises CDRs grafted onto a heterologous framework. 6.The IFNgR2 binding molecule of claim 1, further comprising a labelingagent, an imaging agent, and/or a therapeutic agent.
 7. A nucleic acidsequence encoding the IL2Rg2 binding molecule of claim
 1. 8. Arecombinant viral or non-viral vector comprising a nucleic acid of claim7.
 9. A host cell comprising a nucleic acid of claim
 8. 10. A kitcomprising the IFNgR2 binding molecules of claim
 1. 11. A method fortreating or preventing of an autoimmune or inflammatory disease,disorder, or condition in a mammalian subject by administering to saidsubject a therapeutically effective amount of a IFNgR2 binding moleculeof claim
 1. 12. A pharmaceutically acceptable formulation of an IFNgR2binding molecule of claim
 1. 13. A pharmaceutical formulation comprisingthe viral or non-viral vector of claim 8.